In vitro evolution of an antibody fragment population

所有入选病例均接受全身化疗，方案以EP方案为主。其中57例（77%） 接受胸部放疗，58例（78.4%）接受预防性颅脑照hylactic cranial irradiation，PCI）。通过统计治疗后总有效率（CR+PR）及生存率对所 选病例进行分析。[结果]疗效评价按实体肿瘤的疗效评价标准 （Response EvaluationCriteria In Solid Tumors RECISTl.1）标准进行。 手术组总有效率（CR+PR）：96.8%。非手术组总有效率：93.0%。1、 2、3年生存率为93.2%、85.1%、63.5%。手术组与非手术组1、2、3年 生存率分别为93.54%、87.09%、77.41%，93.02%、83.72%、55.82%， 手术组内观察：pI期、pIIa期、pIIb期1、2、3年生存率分别为：100%、 90.90%、90.90%，100%、88.88%、88.88%，81.81%、63.63%、 54.54%。手术组与非手术组1、2年生存率无明显差异，3年生存率手术 组优于非手术组（P＜0.05）。手术组内分析：pI期~pIIa期生存率无差 异，IIb期生存率明显降低。差异有统计学意义（P＜0.05）。[结论]在早 期小细胞肺癌患者治疗方案的选择中：单纯化、放疗相比，手术治疗联 合化疗或联合化疗+放疗的治疗手段，能明显延长小细胞肺癌患者生存 时间。肿瘤TN分期对早期小细胞肺癌的预后均有明显影响。
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来源数据库：PubMed Journal DOI：10.3390/ijms160511439 关键词：CC chemokine ligand 2 (CCL2); SCLC; blood–brain barrier (BBB); brain metastasis; transendothelial migration; visfatin; 摘要：Small-cell lung cancer (SCLC) is characterized as an aggressive tumor with brain metastasis. Although preventing SCLC metastasis to the brain is immensely important for survival, the molecular mechanisms of SCLC cells penetrating the blood-brain barrier (BBB) are largely unknown. Herein, we present evidence that elevated levels of visfatin in the serum of SCLC patients were associated with brain metastasis, and visfain was increased in NCI-H446 cells, a SCLC cell line, during interacting with human brain microvascular endothelial cells (HBMEC). Using in vitro BBB model, we found that visfatin could promote NCI-H446 cells migration across HBMEC monolayer, while the effect was inhibited by knockdown of visfatin. Furthermore, our findings indicated that CC chemokine ligand 2 (CCL2) was involved in visfatin-mediated NCI-H446 cells transendothelial migtation. Results also showed that the upregulation of CCL2 in the co-culture system was reversed by blockade of visfatin. In particular, visfatin-induced CCL2 was attenuated by specific inhibitor of PI3K/Akt signaling in NCI-H446 cells. Taken together, we demonstrated that visfatin was a prospective target for SCLC metastasis to brain, and understanding the molecular mediators would lead to effective strategies for inhibition of SCLC brain metastasis.

基于转铁蛋白受体(TfR1)的肿瘤与脑部疾病靶向治疗研究进展人转铁蛋白受体（TfR1)在不同组织器官中普遍表达，其主要功能是协助转铁蛋白在细胞和血脑屏障内外转运，维持细胞铁平衡。

在肿瘤细胞中以及血脑屏障中，TfR1的表达水平明显高于正常细胞组织，因此，TfR1被认为是肿瘤靶向治疗和脑部疾病靶向治疗的重要靶点。

基于TfR1靶向治疗的药物载体主要有转铁蛋白（Tf）、抗TfR1抗体、TfR1结合肽，这些生物大分子能与TfR1特异性结合，结合之后可以通过受体介导的跨胞转运机制进入细胞或穿过血脑屏障。

将小分子药与这些载体偶联可以促进许多亲水性的化疗药物或神经治疗药物进入肿瘤细胞或血脑屏障，而许多中枢神经治疗性大分子则主要通过融合蛋白的方式与抗TfR1抗体连接转运进入中枢神经系统。

Abstract：Human TfR1 was universally expressed in different tissues. The major function of TfR1 was to facilitate delivery of transferrin across cells and blood-brain barrier(BBB). As a result, iron homo-stasis was maintained. TfR1 was recognised as a critical target for tumor and brain disease therapy due to its over expression in tumor cells and BBB. In recent years, drug carriers based on TfR1 recognition were developed such as Transferrin (Tf）, anti-TfR1 antibody and TfR1 binding peptide. These carriers bind to TfR1 specifically and enter into cell or BBB through receptor mediated endocytosis. Chemicals conjugated with these carriers can be facilitated to enter into tumor cells and brain tissue. Therapeutic proteins can be engineered to fused with anti-TfR1 antibody and transported across BBB.Key words：TfR1; Tumor target therapy;Brain directed delivery1轉铁蛋白受体（TfR1)简介转铁蛋白受体（TfR1)是一种在不同组织和细胞系中普遍表达的糖蛋白。

转化生长因子-β1(TGF-β1)与肺纤维化研究的进展陈刚;余民浙【摘要】转化生长因子-β1（Transformating Growth Factorbetal，TGF-β1）是一种多功能的细胞因子，是由2条分子量为11Kd有112个氨基酸构成的单链通过二硫键结合而成的分子量为25Kd的多肽。

它在细胞的生长、分化、免疫调节、调节细胞外基质（Extracellular matrix，ECM）合成及损伤后的修复方面发挥着重要的作用。

在哺乳动物中。

TGF—β家族有3个亚型TGF—β1、TGF-β2、TGF—β3，它们通过与相应的受体结合而发挥生物作用。

活化的TGF—β过度表达对肺、【期刊名称】《中国疗养医学》【年(卷),期】2007(016)001【总页数】3页(P3-5)【关键词】转化生长因子-β1;TGF-β2;肺纤维化;细胞因子;免疫调节;细胞外基质;哺乳动物【作者】陈刚;余民浙【作者单位】066104,国家煤矿安全监察局尘肺病康复中心;066000,秦皇岛市海港医院【正文语种】中文【中图分类】R5转化生长因子－β1（Transformating Growth Factor beta1,TGF-β1）是一种多功能的细胞因子，是由 2条分子量为 11Kd有 112个氨基酸构成的单链通过二硫键结合而成的分子量为 25Kd的多肽。

它在细胞的生长、分化、免疫调节、调节细胞外基质（Extracellular matrix,ECM）合成及损伤后的修复方面发挥着重要的作用[1，2]。

在哺乳动物中，TGF-β 家族有3个亚型TGF-β1、TGF-β2、TGF-β3，它们通过与相应的受体结合而发挥生物作用。

活化的 TGF-β 过度表达对肺、肝、肾等组织病理改变的影响非常显著，特别是致纤维化方面。

在体内试验中，TGF-β1对纤维化的作用明确、TGF-β2作用不明确、TGF-β3无作用；然而体外试验发现 TGF-β 的 3个亚型都有促进纤维化的作用。

胸腺素β4研究的进展摘要：胸腺素β4是真核生物细胞中的一种主要的肌动蛋白调节因子，广泛分布于脊椎动物和无脊椎动物的多种组织中及有核细胞中。

尽管其分子水平的作用机制尚不明确，但胸腺素β4却与人类的许多生理及病理过程密切相关。

近年来对胸腺素β4的研究，发现其具有多重生物学功能，与组织再生、重塑、创伤愈合、维持肌动蛋白平衡、肿瘤发病与转移、细胞凋亡、炎症、血管生成、毛囊发育、角膜及心肌修复等密切相关。

随着研究的进一步深入，胸腺素β4在临床上的潜在应用价值将被开发，这对于一些疾病的诊断、治疗及预防均具有重要意义。

文中拟对近年来胸腺素β4在生物学功能上所取得的进展进行综述。

关键词胸腺素β4；肌动蛋白调节因子；伤口愈合；肿瘤转移；瘢痕疙瘩胸腺素(Thymosins)是由胸腺产生的一种淋巴生长因子，由Goldstein和White于1966年首次从胎牛胸腺蛋白提取液中发现，是一组小分子多肽，含有40多种组分[1]。

胸腺素根据等电点不同可分为α、β、γ三类，其中等电点位于5．0～7．0的为β族胸腺素(β-thymosins，Tβ)。

Tβ含40～44个氨基酸，结构高度保守，相对分子质量约为5000，广泛存在于脊椎动物和无脊椎动物中。

迄今已发现的Tβ成员有15个，人体内有3种，即Tβ4、Tβ10和Tβ15。

其中，胸腺素B4分布最广泛、含量最多，占β族胸腺素总量的70-80％ [2,3]。

近年来，Tβ4的生物学功能倍受人们关注，它在许多生理和病理活动中起重要作用。

研究证明，Tβ4具有多重生物学功能，与组织再生、重塑、创伤愈合、维持肌动蛋白平衡、肿瘤发病与转移、细胞凋亡、炎症、血管生成、毛囊发育、角膜及心肌修复等密切相关。

目前，人工合成的Tβ4已大量用于实验中，以探讨其在不同生理和病理活动中的作用机制。

1 Tβ4的结构与分布Tβ4首先于1981年由Low等从胸腺中分离所得，含43个氨基酸，相对分子质量为4921(乙酰化后为4963)，等电点为5.1。

肿瘤坏死因子受体超级家族（tumor necrosis fac⁃tor receptor superfamily，TNFRSF）的死亡受体（death receptor）以及它们的配体在胚胎正常发育及机体免疫和炎症反应过程中扮演了重要角色。

外胚层发育不良受体（ectodysplasin A2receptor，EDA2R）是一个在20年前被鉴定出来的TNFRSF成员（TNFRSF27）［1］，在肿瘤发生、雄激素性脱发等过程中起到重要的作用，但对于该受体作系统性介绍的综述文章尚未见报道。

本文就该受体的研究进展作一系统性的综述，旨在为相关研究提供新的思路。

1EDA2R的蛋白结构和配体1.1EDA2R的蛋白结构EDA2R基因位于人类染色体Xq12，全长约43kb，有6个外显子（GenBank登录号：NG_013271），外胚层发育不良受体EDA2R的研究进展蓝希钳1，2，肖海婷1，2，罗怀容1，2，陈建宁1，2（西南医科大学药学院：1衰老与再生医学实验室，2药理学教研室，四川泸州646000）【摘要】外胚层发育不良受体EDA2R（ectodysplasin A2receptor）是肿瘤坏死因子受体超级家族（tumor necrosis factor recep⁃tor superfamily，TNFRSF）中的一个较新的成员，在发育中的胚胎里有很高的表达，在成年人和动物的多个器官组织中也有表达。

与其它TNFRSF成员不同，尽管EDA2R蛋白在胞内没有死亡结构域（death domain，DD），但它仍可激活NF-κB和JNK通路，并介导细胞的凋亡。

本文广泛回顾了近年来与EDA2R有关的文献，就该蛋白分子的相关研究进展进行综述，以期为与该蛋白相关的分子功能或其介导的相关疾病的研究提供新的思路。

【关键词】EDA2R受体肿瘤坏死因子受体超级家族死亡结构域凋亡【中图分类号】R34文献标志码A doi：10.3969/j.issn.2096-3351.2021.03.018Research progress of ectodysplasin A2receptorLAN Xi-qian1，2，XIAO Hai-ting1，2，LUO Huai-rong1，2，CHEN Jian-ning1，2 1Key Laboratory for Aging and Regenerative Medicine；2Department of Pharmacology，School of Pharmac，South⁃west Medical University，Luzhou646000，Sichuan，China【Abstract】Ectodysplasin A2receptor（EDA2R）is a relatively new member of the tumor necrosis factor re⁃ceptor superfamily（TNFRSF），and it is highly expressed in developing embryos and is also expressed in multiple organs and tissues of adult human and animals.Different from other TNFRSF members，EDA2R protein does not contain the death domain in the intracellular region，but it can still activate the NF-κB and JNK pathways and medi⁃ate cell apoptosis.This article reviews related articles on EDA2R in recent years and related research advances in this protein，in order to provide new ideas for research on molecular functions associated with EDA2R or related dis⁃eases mediated by EDA2R.【Key words】Ectodysplasin A2receptor Tumor necrosis factor receptor superfamily Death domain Apoptosis基金项目：泸州市科技局－西南医科大学联合项目（2018LZXNYD-ZK12）；西南医科大学-泸州市中医医院基地项目（2019-LH005）第一作者简介：蓝希钳，博士。

人表皮聚角蛋白微丝蛋白的提取及其在类风湿关节炎诊断中的应用张丽君　艾脉兴　曾小峰　甘晓丹　史艳萍　宋琴芳　冷晓梅　唐福林 【摘　要】　目的　建立从人表皮提取聚角蛋白微丝蛋白(filaggrin)抗原的方法,检测抗filaggrin 抗体(AFA)在类风湿关节炎(RA)中的分布,探讨AFA对早期RA诊断的价值。

方法　从人表皮细胞中提取并部分纯化filaggrin抗原,经SDS2PA GE后用免疫印迹法检测(Western blot)103例类风湿关节炎(RA)血清和140例对照血清,包括系统性红斑狼疮(SL E)、干燥综合征(SS)、骨关节炎(OA)等。

结果　本研究方法提取的filaggrin能作为AFA免疫印迹检测的抗原。

103例RA病人中AFA敏感性和特异性分别为3519%、9317%,与疾病对照组和正常对照组相比,差异有显著性(P<0105)。

结论　从人表皮提取和部分纯化filaggrin抗原,能满足免疫印迹检测的要求。

用免疫印迹法检测RA 患者的AFA抗体,对RA有较好的敏感性和特异性,可临床用于RA的诊断。

由于基因的变异性,不同个体的filaggrin表达强弱不同。

【关键词】　多聚蛋白质类;关节炎,类风湿;印迹法,蛋白质;抗聚角蛋白微丝蛋白抗体The extraction of f ilaggrin and its signif icance in rheumatoid arthritis ZHA N G L ijun,A I M aixing, ZEN G Xiaof eng,et al.Depart ment of Rheum atology and Im m unology,Peking U nion Medical College Hospital,Beijing100730,China【Abstract】　Objective　To establish the method for extracting the cytokeratin filament2aggregating protein(filaggrin)from human epidermis,to detect the anti2filaggrin antibody(AFA)in rheumatoid arthri2 tis(RA)and to determine the diagnostic value of AFA in RA.Methods　Filaggrin was extracted from hu2 man epidermis and partially purified.AFA in103patients with RA and140controls was detected by West2 ern blotting.R esults　Filaggrin extracted from the epidermis could be used as the antigen in AFA test by Western blotting.AFA test resulted in diagnosis of3519%of103RA samples,with a specificity of 9317%.The result was significantly higher compared with patients′and normal controls(P<0105).Con2 clusion　Filaggrin extracted from the human e pidermis could be used in Western blotting.AFA has diag2 nostic value in RA.The individuality expresses heterogeneous levels of filaggrin for the gene variation.【K ey w ords】　Polyproteins;Arthritis,rheumatoid;Blotting,Western;Antifliaggrin antilbody 人表皮提取的聚角蛋白微丝蛋白(cytokeratin filament aggregating protein,filaggrin)是一种居间纤维。

介导性shRNA能抑制肺癌细胞中livin沉默基因的表达从而促进SGC-7901细胞凋亡背景—由于肿瘤细胞抑制凋亡增殖,特定凋亡的抑制因素会对于发展新的治疗策略提供一个合理途径。

Livin是一种凋亡抑制蛋白家族成员，在多种恶性肿瘤的表达中具有意义。

但是, 在有关胃癌方面没有可利用的数据。

在本研究中,我们发现livin基因在人类胃癌中的表达并调查了介导的shRNA能抑制肺癌细胞中livin沉默基因的表达，从而促进SGC-7901细胞凋亡。

方法—mRNA及蛋白质livin基因的表达用逆转录聚合酶链反应技术及西方吸干化验进行了分析。

小干扰RNA真核表达载体具体到livin基因采用基因重组、测序核酸。

然后用Lipofectamin2000转染进入SGC-7901细胞。

逆转录聚合酶链反应技术和西方吸干化验用来验证的livin基因在SGC-7901细胞中使沉默基因生效。

所得到的稳定的复制品用G418来筛选。

细胞凋亡用应用流式细胞仪(FCM)来评估。

细胞生长状态和5-FU的50%抑制浓度(IC50)和顺铂都由MTT比色法来决定。

结果—livin mRNA和蛋白质的表达检测40例中有19例(47.5%)有胃癌和SGC-7901细胞。

没有livin基因表达的是在肿瘤邻近组织和良性胃溃疡病灶。

相关发现在livin基因的表达和肿瘤的微小分化和淋巴结转移一样(P < 0.05)。

4个小干扰RNA真核表达矢量具体到基因重组的livin基因建立。

其中之一,能有效地减少livin基因的表达,抑制基因不少于70%(P < 0.01)。

重组的质粒被提取和转染到胃癌细胞。

G418筛选所得到的稳定的复制品被放大讲究。

当livin基因沉默,胃癌细胞的生殖活动明显低于对照组(P < 0.05)。

研究还表明,IC50上的5-Fu 和顺铂在胃癌细胞的治疗上是通过shRNA减少以及刺激这些细胞(5-Fu proapoptotic和顺铂)(P < 0.01)。

荧光纳米级显微镜揭示HIV-1病毒成熟过程中表面蛋白结构重排泛瑞翻译人类免疫缺陷病毒I型（Human immunodeficiency virus type 1，HIV-1）从宿主细胞出芽后依然是未成熟的粒子，其主要结构蛋白Gag经过后续的酶解后才能形成成熟的病毒粒子且具有侵染能力。

10月26日的科学杂志刊发了德国海德堡大学一个团队的研究文章，他们利用超高倍成像技术直观观察了HIV病毒粒子成熟的关键步骤。

Chojnacki等通过利用受激发射湮灭超高分辨率荧光显微镜观察HIV-1病毒外层包膜糖蛋白在病毒颗粒上的分布，发现成熟过程诱发的包膜蛋白聚集依赖于包膜蛋白尾端和Gag蛋白互作。

他们把糖蛋白在表面的聚集程度和病毒的侵染效率关联分析后发现，病毒内部和外部有着巧妙的耦合作用：内部蛋白结构的酶解后结构重排促使病毒表面结构改变，为侵染做准备。

Chojnacki等认为Gag酶解诱发的稀少的包膜蛋白二聚体在病毒表面聚集可能是HIV-1成熟的一个基本方面。

这一运用直观的高倍成像技术揭示的HIV成熟的过程和机制，有利于改善人们研发临床HIV疫苗的操作和技术，具有重要的理论和指导意义。

Chojnacki, J., T. Staudt, et al. (2012). "Maturation-Dependent HIV-1 Surface Protein Redistribution Revealed by Fluorescence Nanoscopy." Science 338(6106): 524-528.Human immunodeficiency virus type 1 (HIV-1) buds from the cell as an immature particle requiring subsequent proteolysis of the main structural polyprotein Gag for morphological maturation and infectivity. Visualization of the viral envelope (Env) glycoprotein distribution on the surface of individual HIV-1 particles with stimulated emission depletion (STED) superresolution fluorescence microscopy revealed maturation-induced clustering of Env proteins that depended on the Gag-interacting Env tail. Correlation of Env surface clustering with the viral entry efficiency revealed coupling between the viral interior and exterior: Rearrangements of the inner protein lattice facilitated the alteration of the virus surface in preparation for productive entry. We propose that Gag proteolysis-dependent clustering of the sparse Env trimers on the viral surface may be an essential aspect of HIV-1 maturation.。

Original ArticleSignal peptide replacements enhance expression and secretion of hepatitis C virus envelope glycoproteinsBo Wen1,2†,Yao Deng2†,Jie Guan2,Weizheng Yan2,Yue Wang2,Wenjie Tan1,2,and Jimin Gao1*1Wenzhou Medical College,Wenzhou325000,China2State Key Laboratory for Molecular Virology and Genetic Engineering,National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing100052,China†These authors contributed equally to this work.*Correspondence address.Tel/Fax:þ86-10-63552140;E-mail:tanwj28@(W.T.);Tel/Fax:þ86-577-86689748;E-mail:jimingao@ (J.G.)A large number of researches focused on glycoproteins E1 and E2of hepatitis C virus(HCV)aimed at the develop-ment of anti-HCV vaccines and inhibitors.Enhancement of E1/E2expression and secretion is critical for the charac-terization of these glycoproteins and thus for subunit vaccine development.In this study,we designed and syn-thesized three signal peptide sequences based on online programs SignalP,TargetP,and PSORT,then removed and replaced the signal peptide preceding E1/E2by over-lapping the polymerase chain reaction method.We assessed the effect of this alteration on E1/E2expression and secretion in mammalian cells,using western blot analysis,dot blot,and Galanthus nivalis agglutinin lectin capture enzyme immunoassay.Replacing the peptides pre-ceding E1and E2with the signal peptides of the tissue plasminogen activator and Gaussia luciferase resulted in maximum enhancement of E1/E2expression and secretion of E1in mammalian cells,without altering glycosylation. Such an advance would help to facilitate both the research of E1/E2biology and the development of an effective HCV subunit vaccine.The strategy used in this study could be applied to the expression and production of other glyco-proteins in mammalian cell line-based systems. Keywords hepatitis C virus;signal peptide;envelope glycoprotein;expression;secretionReceived:July13,2010Accepted:October26,2010 IntroductionHepatitis C virus(HCV)is a small,enveloped, positive-stranded RNA virus that belongs to the Hepacivirus genus in the Flaviviridae family[1].HCV infection often leads to chronic hepatitis,liver cirrhosis,and hepatocellular carcinoma.Despite its clinical significance,the functional characterization of HCV glycoproteins,and thus vaccine development,has been hindered by a paucity of knowledge relating to the major structural glycoproteins E1/E2[1,2]. The HCV genome encodes a single polyprotein that is processed by cellular and viral proteases to generate10 polypeptides[3].The HCV virion is thought to have a diameter of 50nm[4].It is composed of a nucleocapsid surrounded by a host cell-derived membrane envelope that contains the viral glycoproteins E1(polyprotein residues 192–383)and E2(residues384–746)[5].Because they are exposed at the surface of the virion,the envelope pro-teins are the target of neutralizing antibodies.Therefore,E1 and E2represent the most immunologically significant HCV antigens.For this reason,E1and E2were the main focus of HCV vaccine development[4,6].HCV glycoproteins are type I membrane proteins with a C-terminal transmembrane domain(TMD)anchored in the virion phospholipid envelope.In their functional form,E1 and E2are thought to form a non-covalent heterodimer,and their TMDs are essential for heterodimerization[5,7].The ectodomains of the HCV envelope glycoproteins E1and E2 are highly modified by N-linked glycans,which have been shown to play a major role in protein folding,virus entry, and protection against neutralizing antibodies[5,7–11]. The enhancement of E1/E2expression and secretion is vital for the characterization of E1/E2structure and func-tion and for the development of an effective E1/E2-based subunit vaccine[4–7].To our knowledge,native E1/E2 expressed in mammalian cells is unsuitable for this purpose.However,some studies have shown that the optimization of signal peptide may enhance the levels of expression and secretion of these glycoproteins[12–14]. Similar strategies also have been described in researches of HIV and some other viruses[15–19].Acta Biochim Biophys Sin2011,43:96–102|ªThe Author2010.Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology,Shanghai Institutes for Biological Sciences,Chinese Academy of Sciences.DOI:10.1093/abbs/gmq117.Advance Access Publication31December2010Acta Biochim Biophys Sin(2011)|Volume43|Issue2|Page96 by guest on January 29, 2011 Downloaded fromIn this study,we constructed several plasmids encoding E1and E2to assess the effect of the replacement of the signal peptide sequences located upstream of the E1and E2genes.Our results suggested that replacing the signal sequence preceding the E1and E2genes with three signal peptide sequences significantly enhanced expression and secretion of eE1(a secreted form of E1)and eE2(a secreted form of E2)in mammalian cells.Such modifi-cations represent a significant advance in the technique available to investigate the E1and E2structure and their function and will facilitate the development of an HCV entry inhibitor and/or effective vaccine.Materials and MethodsCell culture and plasmid constructionHuman embryo kidney293T cells were grown in Dulbecco’s modified essential medium(Invitrogen, Carlsbad,USA)supplemented with10%fetal bovine serum(FBS)and100U/ml penicillin/streptomycin.Cells were routinely maintained at378C with5%CO2.Wild-type HCV1b E1and E2genes were amplified from the plasmid as described previously[20].To replace their signal peptide regions,the E1and E2genes minus the signal peptide were amplified by polymerase chain reaction(PCR)using long upstream primers.These primers were used to add the signal peptide of self-designed [21,22],tissue plasminogen activator(tPA)[23,24]or Gaussia luciferase(Gluc)[25](Table1),to the E1and E2 genes.Each of these primers contained15nucleotides of the E1or E2sequence spanning the signal peptidase clea-vage site.An Eco RV site and a Bst EII site were added in those primes for clone construction.To facilitate down-stream purification,a Bam HI restriction site and a DNA sequence encoding a His6-tag peptide were synthesized and added to the30end of the E1and E2sequences by PCR (Fig.1).Standard recombinant DNA techniques were used to generate all constructs.After purification,fragments con-taining the desired signal sequence were digested with Eco RV/Bam HI and then cloned into the same sites in the pVRC8301vector.The general scheme of plasmid con-struction is shown in Fig.1and Table2.Cell transfectionTransient transfections were performed by plating2Â105cells/well in a12-well plate12h prior to initiation of the study.When cells were 80–90%confluent,the experiments were performed.On the day of transfection,the original growth medium was replaced with a serum-free medium and DNA transfection was performed using the FUGENE HD Transfection Reagent(Roche,Basel, Switzerland)according to the standard protocols with slight modifications.Briefly,DNA solution containing1m g of pVRC-E1/E2was diluted in50m l of Opti-MEM;and the ratio of transfection reagent:DNA was6:2.Mixtures were incubated at room temperature for15min.Culture medium in12-well plates was removed and thecells were washed with Opti-MEM.A total of900m l of Table1Prediction results for different signal peptidesSignalpeptide(Sp)Sequence and cleavesite prediction aExtracellular rate ink-NN predictionSpþE1(%)SpþE2(%)E1M G C S F S I F L L AL L S C L T T P A SA ...44.4E2M V G N W A K V L IV M L L F A G V D G...55.6Self-design(sd)MD A M K V L L L VF V S P S Q V TG ...66.777.8Gluc M G V K V L F A L IC I A V A E V T G...b66.766.7tPA M D A M K R G L CC V L L L C G A V FV D S V T G ...66.766.7a Protein cleavage sites are indicated by .b The SignalP-NN result is AVA EV,but the SignalP-HMM result isVTG ,and the SIG-Pred result conforms toHMM.Figure1Schematic representation of HCV E1(A)and E2(B)expression vectors Arrows indicate signal peptide cleavage sites.Signal peptide replacements enhance HCV envelope glycoprotein expression and secretionActa Biochim Biophys Sin(2011)|Volume43|Issue2|Page97by guest on January 29, 2011Downloaded fromfresh serum-free Opti-MEM and50m l of transfection mixture were mixed and added to each well.After48h of incubation,the culture medium was collected and cell pellets were separated by centrifugation at1200g for 5min.Pellets were washed with PBS and then lysed in 200m l of lysis buffer for30min on ice and centrifuged at 14,500g for5min;the clarified cell lysate was collected. Clarified lysate and culture medium were used for expression analysis[20].Identical cDNA amount of pVRC was transfected as a control.PNGase F digestion for analysis of E1and E2 glycosylationsCell lysates and culture medium were diluted in sodium dodecyl sulfate–polyacrylamide gel electrophoresis(SDS–PAGE)loading buffer and heated at1008C for10min. PNGase F(NEB)digestion was performed in1ÂG7 buffer,with1%NP-40at378C for1h.Enzyme-treated samples were analyzed by resolving on10–13%SDS–PAGE gels and western blot analysis.Western blot analysisAfter separation by SDS–PAGE(10–13%),proteins were electroblotted onto a nitrocellulose membrane(Whatman, Florham Park,USA).Monoclonal antibodies to E1(A4)or E2(AP33)(GeneTech,Redwood,USA)were used as the primary antibody with appropriate dilution(1:1500).The horseradish peroxidase(HRP)-conjugated AffiniPure goat anti-rabbit immunoglobulin G(IgG;1:5000dilution; Sigma,St Louis,USA)was used as the secondary anti-body.Proteins were revealed on Thermo film in a dark room using the SuperSignal West Pico Chemiluminescent Substrate(Pierce,Rockford,USA)as recommended by the manufacturer.Dot blot assayA nitrocellulose membrane was pre-wetted with PBS and the Dot blot apparatus(Bio-Rad,Hercules,USA)was assembled according to the manufacturer’s introductions. Culture media and diluted cell lysates were added to wells and vacuum was applied to load products onto the mem-brane.Wells were washed twice with200m l of PBS in a low vacuum.The apparatus was disassembled and the membranes were blocked with blocking buffer(PBSþ3% FBS).After extensive washing,the primary antibody, HCV-infected human serum diluted1:200in FBS buffer, was added.Then,the secondary antibody,HRP-conjugated goat anti-mouse IgG(Sigma)diluted1:5000,was added. Galanthus nivalis agglutinin lectin capture EIA Galanthus nivalis agglutinin(GNA)lectin at1m g/ml (Sigma)was used to coat enzyme immunoassay/radio-immunoassay(EIA/RIA)plates(Costar Group,Cambridge, USA)overnight at48C.After being washed with PBS, plates were blocked with5%milk powder,and then super-natants or cell lysates were added for binding at room temperature.Plates were washed with a washing buffer (PBSþ0.05%Tween-20)four times,then100m l of primary antibody monoclonal antibody(MAbs)A4or AP33,dilution at1:1500)was added,followed by incubat-ing for90min at378C.Unbound antibody was washed off with the washing buffer.HRP-conjugated goat anti-mouse IgG(secondary antibody,100m l)was added to each well at1:5000dilution in5%milk powder and incubated for 45min at378C.Finally developed with a tetramethylbenzi-dine substrate and absorbance values at450nm were determined.ResultsReplacement of the sequence encoding the signal peptide of E1and E2The online programs SignalP,TargetP,and PSORT were used to determine cleavage sites and the expression localiz-ation of various signal peptides[21,22].Our goal was to select the peptide that could facilitate higher extracellular expression of the E1and E2proteins compared with the native signal peptide.E1and E2genes were modified to enable a series of translational fusions with different signalTable2Plasmids used in this studyPlasmid Relevant characteristicpVRC8301Efficient expression vector in eukaryotic cellsE1pVRC-E101pVRC8301derivative containing the wild-typeHCV(1b)E1(amino acids170–311)pVRC-E102pVRC-E101derivative with the signal peptidesequence replaced with a self-designed signalsequencepVRC-E103pVRC-E101derivative with the signal peptidesequence replaced by that of tPApVRC-E103pVRC-E101derivative with the signal peptidesequence replaced by that of GlucE2pVRC-E201pVRC8301derivative containing the wild-typeHCV(1b)E2(amino acids364–661)pVRC-E202pVRC-E201derivative with the signal peptidesequence replaced with a self-designed signalsequencepVRC-E203pVRC-E201derivative with the signal peptidesequence replaced by that of tPApVRC-E203pVRC-E201derivative with the signal peptidesequence replaced by that of GlucSignal peptide replacements enhance HCV envelope glycoprotein expression and secretionActa Biochim Biophys Sin(2011)|Volume43|Issue2|Page98 by guest on January 29, 2011 Downloaded frompeptide-encoding regions.An Eco RV site and a Bst EII site were inserted at each end of the signal peptide-encoding region(Fig.1).The Bst EII insertion created a mutation in the C-terminal of each signal peptide,in which the terminal amino acid was replaced with V-T-G(Fig.1).Software analysis indicated that these replacements did not alter the cleavage site of E1/E2(Table1).For expression,the frag-ment obtained by digesting each PCR product with Eco RV/Bam HI was inserted at the same sites in the pVRC8301vector.Plasmids containing the wild-type signal peptide sequence were named pVRC-E101and pVRC-E201;plasmids containing the computer-designed signal peptide sequence were named pVRC-E102and pVRC-E202;plasmids containing the Gluc signal peptide sequence were named pVRC-E103and pVRC-E203;and plasmids containing the tPA signal peptide sequence were named pVRC-E104and pVRC-E204(Table2).In each case,successful insertion was confirmed by sequencing. Effects of signal peptide replacement on intracellular expression of E1and E2The signal peptide sequence of the HCV E1/E2glyco-proteins was substituted with three heterologous signal peptide sequences.Intracellular and extracellular expression levels of E1and E2proteins were assessed using a mono-clonal antibody specific for either E1or pared with the control(pVRC-E101/pVRC-E201),all of the clones (pVRC-E102/pVRC-E202,pVRC-E103/pVRC-E203,and pVRC-E104/pVRC-E204)successfully expressed the E1or E2glycoproteins.HCV E1expressed in untreated cells had an apparent molecular mass of33kDa and the molecular weight(MW)was reduced to18kDa after deglycosylation, and the MW of E2is60–70kDa in nature and31kDa after deglycosylation[3].Our results showed that the MW of each modified signal peptide attached to E1and E2did not differ significantly from the wild type(Fig.2).Furthermore, after digestion with N-glycosidase F,the MWs of E1and E2with the modified signal peptide were not significantly different from that of wild type,suggesting a pattern of identical glycosylation(Fig.3).Native E1glycoprotein was detected with the least expression,despite its expression conditions was identical with others.Only by increasing the loading quantity to 30m g(six times of the loading quantity than that of other samples),the band of native E1glycoprotein can be detected(Fig.2).However,band intensity remained notice-ably lower than any of the modified E1proteins.Thus,pro-duction of E1with a wild-type signal sequence was particularly low,at least less than one-sixth,that of E1 with either the tPA or Gluc signal peptide.Effect of signal peptide replacement on secretionof E1/E2After confirming the intracellular expression of E1/E2with the replaced signal peptide,we assessed the extracellular concentrations of the E1and E2proteins.As predicted by software,the ratios of secretion were different among the various signal peptides.To assess the true expression level of the modified E1and E2glycoproteins,we chose methods that could enrich the non-denature productions for more sen-sitive detection,such as dot blot and GNA capture EIA, both the semi-quantitative methods.They were used to detect glycoproteins E1and E2in all cell lysates and culture media in parallel.Results from these two methods showed no obvious disparities(Fig.4).The expression level of the E1native signal peptide was particularly low,and three of the modified signal peptides,particularly tPA and Gluc, showed significantly increased expression than thenative Figure2Western blot analysis of E1/E2produced by293T transfected with different plasmids Cell lysate samples(L,5m g/sample)and supernatants(S,20m g/sample)were electrophoresed separately.All samples were collected after48h cultivation with non-BSA opti-MEM.The blot was stripped and re-probed with b-actin.Asterisk denotes that the loading volume of this sample was six times greater(30m g)than that of other lysate samples.293T cells transfected without a plasmid was used as a control(mock).Signal peptide replacements enhance HCV envelope glycoprotein expression and secretionActa Biochim Biophys Sin(2011)|Volume43|Issue2|Page99 by guest on January 29, 2011 Downloaded fromsignal peptide.Such distinction was not so obvious in E2,but the expression level of HCV E2glycoprotein with a native signal peptide was still lower than that of E2modified with any of the exogenous signal peptides.Otherwise,the expression level of pVRC-E102/pVRC-E202,whose signal peptide was predicted to have the greatest secretion ability,was lower than the sample with tPA or Gluc signal peptides.Furthermore,modified with the tPA or Gluc signal peptides,neither the expression nor the secretion levels of glyco-proteins showed any marked differences (Fig.4).In our experiment,E2could be more easily detected than E1.This might be caused by different primary antibody,or because E1/E2were glycosylated at different levels leading to dis-tinct GNA-binding ability.DiscussionMore than 120million people worldwide are chronically infected with HCV,making HCV infection to be the leading cause of liver transplantation in developed countries.Treatment options are limited,and the efficacy depends on both the infecting strain and the initial viral load [1].During the translation of HCV,the nascent E1and E2polypeptides are targeted to the host endoplasmic reticulum membrane for modification by N-linked glycosylation [5].E1and E2are released from the polyprotein through cleavage by a host signal peptidase [3]and are anchored in the viral lipid envel-ope as a heterodimer,which plays a major role in the HCV entry [7,11].Deletion of the TMDs of E1and E2results in the secretion of these truncated forms of HCV glycoproteins into the extracellular medium.E2antigen in itssecretedFigure 4Analysis of the production levels of E1and E2glycoproteins containing altered signal peptides Human embryo kidney 293T cells contained the following plasmids were indicated,and 293T cells transfected without a plasmid was used as a control (mock).Cell lysates (L)and supernatants (S)were collected 48h after transfection.(A)and (B)represent the dot blot results of E1and E2glycoproteins detected using HCV-infected human serum as the primary antibody.GNA lectin capture EIA data are shown in (C)and (D).Data obtained from both the dot blot and EIA methods showed no markeddiscrepancies.Figure 3Different signal peptide have no apparently effect on the expression of E1and E2in 293T cells 293T cells contain pVRC-E101/104and pVRC-E201/204plasmids were collected 48h after transfection.Cell lysate (L)and supernatant (S)of each samples were treated with PNGase F (W)or left untreated (W/O).After being separated by SDS–PAGE (E1in 13%and E2in 10%acrylamide)under reducing conditions,E1and E2were immunoprecipitated with monoclonal antibodies A4and AP33.(A)In the same loading quantity (20m g),bands detected from E101(arrow indict)were much fainter than those of E104,but their distributions were the same under the similar treatment.(B)The distinction of band grayscale between E201and E204is not obvious,but the MW and distribution of their bands are identical under the same treatment.Signal peptide replacements enhance HCV envelope glycoprotein expression and secretionActa Biochim Biophys Sin (2011)|Volume 43|Issue 2|Page 100by guest on January 29, 2011 Downloaded fromform leads to an increased humoral response in mice[26]. HCV E1and E2are primary determinants of entry and pathogenicity.HCV E2glycoproteins are involved in recep-tor binding,virus–cell fusion,and entry into host cells[11]. However,its role in membrane fusion and immune evasion remains uncharacterized.The function of E1is unknown; however,it is a target for neutralizing antibodies and its association with E2is essential for viral entry[11]. Increasing knowledge of the nature and the function of HCV E1and E2glycoproteins help to develop antiviral drugs and vaccine candidates.The production of large quantities of functional and secreted E1and E2proteins will allow us to perform compre-hensive biochemical and biophysical analysis,which will lead to the development of a vaccine that is effective against HCV infection.In this study,we developed a novel expression system for producing the secreted form of E1(eE1)and E2 (eE2)ectodomains from mammalian cells via a signal peptide sequence replacement strategy and performed a comprehen-sive biochemical characterization.These data enhance our understanding of HCV envelope glycoproteins and may assist in the design of HCV vaccines and entry inhibitors. Our data showed that the replacement of the signal peptide sequences located at the upstream of E1and E2 genes altered their secretion and expression levels,and most importantly,the replacement did not affect glycosyla-tion.Furthermore,replacing them with tPA and Gluc resulted in the greatest increases at expression and secretion levels of eE1and eE2in mammalian cells.We believed that the strategy used in this study could be applied to the expression and production of other glycoproteins in mam-malian cell line-based systems.The next challenge is to use our novel strategy to gener-ate large quantities of HCV E1and E2envelope glyco-proteins.This study will allow us to elucidate their structural biology and the mechanism(s)involved in the interactions between these glycoproteins and other cellular factors,eventually to facilitate the development of an HCV vaccine and/or entry inhibitor. AcknowledgementsThe authors thank Dr Gary Nabel(Vaccine Research Center,National Institute of Allergy and Infectious Diseases,National Institutes of Health,Bethesda,USA)for the pVRC plasmid and Dr Jean Dubuisson(Universite´Lille Nord de France,CNRS-UMR8161,Institut Pasteur de Lille,Lille,France)for the A4antibody.FundingThis work was supported by the grants from the Hi-Tech Research and Development Program of China(2007AA02Z455,2007AA02Z157)and the State KeyLaboratory for Molecular Virology and Genetic Engineering. 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1周博达1 侯超1 综述 王月丹1北京大学医学部基础医学院 100083Email: wangyuedan@摘要: 血管内皮生长因子通过与其受体结合发挥生物学作用，促进内皮细胞有丝分裂及其增殖分化、迁移，导致血管新生，形成血管翳，在类风湿关节炎的病理过程中发挥关键的作用。

对抗血管内皮生长因子及其受体，从而阻止新生血管的形成为治愈类风湿关节炎提供了新的途径。

关键词：类风湿性关节炎；血管内皮生长因子；受体；类风湿关节炎(rheumatoid arthritis, RA)是一种严重的自身免疫功能障碍性疾病，在世界范围内的平均患病率约1%，而我国约有400 万RA患者。

其临床表现以反复发作的多发性小关节炎和不同程度侵蚀性改变及进行性强硬和畸形为主，并有骨和骨骼肌萎缩，是一种致残率较高的疾病。

RA的病理特征为关节滑膜慢性炎症、软骨吸收、骨质破坏和骨纤维化，其中一个重要的病理生理过程是新生血管翳的形成，而新生血管的生成是产生和维持RA血管翳的重要标志。

在RA发展过程中，滑膜组织中存在大量的血管,出现了血源性细胞的浸润、增殖和水肿，在血管内皮细胞里层也出现了对细胞因子等作用的靶点，同时内皮细胞对这些因子的反应可以维持和促进RA病理过程的发展，特别是新血管的生成[1]。

近年来，人们已经认识到血管生成在RA的侵蚀和破坏过程中发挥了重要作用。

中断血管生成在类风湿性关节炎的治疗中有重要的意义。

RA患者的关节滑膜中，存在一些生长因子、细胞因子和化学增活素从而影响血管的生成，许多生物活性物质还能通过上调血管生成刺激素的表达从而发挥间接作用。

其中，血管内皮生长因子及其受体在血管新生过程中起着重要的作用。

一、关于血管内皮生长因子及其受体血管内皮生长因子(vascular endothelial growth factor，VEGF)，又称为血管通透性因子(vascular permeability factor, VPF)，于1989年由Ferrara等发现，属于血小板源生长因子(platelet-derived growth factor, PDGF) 家族成员，是一种相对分子量在34000-46000的同源二聚体糖蛋白，由两个亚基通过二硫键结合，其热稳定性高，序列高度保守。

DOI:10.12280/gjszjk.20200730谢奇君，李欣，赵纯，凌秀凤△【摘要】辅助生殖技术（ART）已被广泛应用于不孕症的治疗，包括人工授精（AI）和体外受精-胚胎移植（IVF-ET），及其相关衍生技术如胞浆内单精子注射（ICSI）、胚胎植入前遗传学筛查（PGS）、卵母细胞体外成熟（IVM）、胚胎辅助孵化（AH）等技术。

随着ART的普及和妊娠率的逐步提高，其带来的并发症及其安全性也越来越受到重视。

能够及时发现和处理并发症，可以最大限度地使不孕症患者获得安全优质的妊娠结局。

目前，ART相关并发症发生的原因仍存在争议，有部分研究认为是由于ART导致的多胎妊娠或不孕症病因，也有研究认为是由于ART本身。

故对近年来相关文献进行分析，总结ART相关并发症的最新研究进展。

【关键词】生殖技术，辅助；并发症；不育，女（雌）性；体外受精；胚胎移植Research Progress of Complications Related to Assisted Reproductive Technology XIE Qi-jun,LI Xin,ZHAO Chun,LING Xiu-feng.Reproductive Center,Women′s Hospital of Nanjing Medical University(NanjingMaternity and Child Health Care Hospital),Nanjing210004,ChinaCorresponding author:LING Xiu-feng,E-mail:************************【Abstract】Assisted reproductive technology(ART)has been widely used in the treatment of infertility,including artificial insemination(AI)and in vitro fertilization-embryo transfer(IVF-ET),and many derivativetechniques such as intracytoplasmic sperm injection(ICSI),preimplantation genetic screening(PGS),in vitromaturation(IVM),and assisted hatching(AH).With the popularity of ART and the gradual improvement ofpregnancy rate,more and more attention has been paid to the complications of ART and the safety of ART.Detection and treatment of complications in time can maximize the safety and high quality of pregnancy,and thegood outcomes for patients.At present,the causes of ART-related complications are still controversial.Somestudies believe that the increased rate of multiple pregnancy caused by ART or the factors related to infertility arethe main causes.However,other studies believe that the factors of ART itself cannot be overlooked.In this review,we analyzed the complications related to ART and the causes.【Keywords】Reproductive techniques，assisted；Complications；Infertility，female；Fertilization in vitro；Embryo transfer（ＪＩｎｔＲｅｐｒｏｄＨｅａｌｔｈ蛐ＦａｍＰｌａｎ，2021，40：204-208）·综述·基金项目：国家自然科学基金（81871210）作者单位：210004南京医科大学附属妇产医院（南京市妇幼保健院）生殖中心通信作者：凌秀凤，E-mail：************************△审校者辅助生殖技术（assisted reproductive technology，ART）是一种应用各种技术处理精子或卵子，以帮助不孕症夫妇实现生育的方法，包括人工授精（artificial insemination，AI）、体外受精-胚胎移植（invitro fertilization-embryo transfer，IVF-ET）及相关技术，如胞浆内单精子注射（intracytoplasmic sperminjection，ICSI）、胚胎植入前遗传学筛查（preimplantation genetic screening，PGS）、卵母细胞体外成熟（in vitro maturation，IVM）、胚胎辅助孵化技术（assisted hatching，AH）和卵母细胞玻璃化冷冻技术等。

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As2 O3 通过抑制肿瘤细胞增殖、诱导肿瘤细胞凋 亡及分化、阻止肿瘤血管新生、提高机体免疫功能等机 制发挥抗肿瘤作用，但其作用机制极其复杂，除上述机 制外，可能还存在着其他途径。因此加强对 As2 O3 抗 肿瘤机制的研究，从新的角度探索其可能的作用机制， 将为砷剂的开发及利用提供科学的理论依据，亦可作 为临床治疗胰腺癌的新策略，为改善胰腺癌病人的生 活质量和延长生命带来希望。
刊） ： 1－2．
［4］ 朱应昌，包仕延，邱志东，等． 亚砷酸对人胰腺癌 PANC－1 细胞增

生物技术进展2022年第12卷第3期358~365Current BiotechnologyISSN 2095‑2341进展评述Reviews抗体药物的发展与应用杨懿祺，张志高，游小龙，张婧，林冠峰，吴英松*南方医科大学检验与生物技术学院，广州510515摘要：早期抗体药物是鼠源单克隆抗体，存在免疫原性强、半衰期短等问题。

历经数十年的发展，抗体药物从最初的鼠源单抗，逐步发展为人鼠嵌合抗体、人源化抗体及全人源化抗体。

通过片段重组、位点修饰、药物偶联等方法，科研人员研发了包括抗体融合蛋白、抗体偶联药物、双特异性抗体、小分子抗体片段等形式多样的抗体药物。

抗体药物在恶性肿瘤、自身免疫病、感染性疾病的治疗上发挥重要作用。

通过对抗体药物人源化历程，不同类型的抗体结构和特点，以及抗体药物在新型冠状病毒肺炎治疗中的应用进行综述，并对抗体药物的发展前景进行展望，以期为我国抗体药物的研发提供参考。

关键词：抗体药物；人源化；抗体融合蛋白；抗体偶联药物；双特异性抗体；小分子抗体片段；新型冠状病毒肺炎DOI ：10.19586/j.2095⁃2341.2021.0173中图分类号：Q511，R97文献标志码：ADevelopment and Application of Monoclonal Antibody -based DrugYANG Yiqi ，ZHANG Zhigao ，YOU Xiaolong ，ZHANG Jing ，LIN Guanfeng ，WU Yingsong *School of Laboratory and Biotechnology ，Southern Medical University ，Guangzhou 510515，ChinaAbstract ：Monoclonal antibody -based drug at first were mouse -derived antibodies ，with the problem of high immunogenicity and short serum half -lives.After the development of several decades ，monoclonal antibody -based drug developed from murine antibod‐ies to chimeric antibodies ，humanised antibodies and full human antibodies.By recombining antibody fragments ，modifying sites and conjugating drugs ，researchers have developed different kinds of monoclonal antibody -based drug ，which included antibody fusion protein ，antibody -drug conjugates ，bispecific antibodies and antibody fragments.All of them played important roles in the treatment of malignant tumour ，autoimmune diseases and infectious diseases.This article summarized the humanization course of monoclonal antibody -based drug ，the types of antibodies'construction and characteristics ，the application of monoclonal anti‐body -based drug in the treatment of coronavirus disease 2019（COVID -19），and offered a brief future perspective for monoclonal antibody -based drug.The paper was expected to provide references for the development of antibody -based drugs.Key words ：monoclonal antibody -based drug ；humanization ；antibody fusion protein ；antibody -drug conjugates ；bispecificantibodies ；antibody fragments ；COVID -19抗体（antibody ）又称免疫球蛋白，是由B 淋巴细胞接受刺激后产生的糖蛋白，结构呈“Y ”字型，具有特异性结合抗原的功能，在人体抵御外界感染性病原体中发挥重要作用。

松萝提取物对胶原诱导性关节炎大鼠高迁移率族蛋白B1介导的细胞自噬及炎症因子的影响作者：文红安阳张军徐晖陆道敏曹跃朋宁乔怡王莹来源：《风湿病与关节炎》2023年第09期【摘要】目的：研究松萝提取物对胶原诱导性关节炎（CIA）大鼠滑膜细胞中凋亡相关因子的影响，进一步探讨松萝提取物对CIA大鼠关节炎症及高迁移率族蛋白B1（HMGB1）介导的细胞自噬的影响。

方法：将54只Wistar雌性大鼠随机分为空白对照组，模型对照组，羟氯喹组，松萝提取物低、中、高剂量组，每组9只。

松萝提取物低、中、高剂量组给药剂量分别为2 mg·kg-1、6mg·kg-1、54 mg·kg-1，羟氯喹组给药剂量为36 mg·kg-1。

建立CIA大鼠模型后，连续灌胃给药28 d。

ELISA法检测血清中HMGB1、白细胞介素-1β（IL-1β）、肿瘤坏死因子-α（TNF-α）的浓度，RT-PCR检测HMGB1、Beclin1、PI3K C3 mRNA的表达，Western Blot法检测HMGB1、Beclin1、PI3K C3蛋白表达。

结果：与空白对照组比较，各模型组HMGB1、TNF-α、IL-1β含量均增高，HMGB1、PI3K C3蛋白表达上升，Beclin1蛋白表达下降，差异有统计学意义（P < 0.05）；与模型对照组比较，松萝提取物中、高剂量组和羟氯喹组HMGB1、TNF-α、IL-1β含量均降低，且HMGB1、PI3K C3蛋白表达下降，Beclin1蛋白表达上升，差异有统计学意义（P < 0.05）。

结论：松萝提取物通过上调Beclin1蛋白，下调HMGB1、PI3KC3相关蛋白的表达，调节细胞自噬，降低CIA大鼠血清中HMGB1、IL-1β、TNF-α浓度，减轻关节炎症。

【关键词】类风湿关节炎；松萝提取物；胶原诱导性关节炎；高迁移率族蛋白B1；关节炎症；自噬；大鼠Effects of Usnea Extract on Cell Autophagy and Inflammatory Factors Mediated by High Mobility Group Protein B1 in Collagen Induced Arthritis RatsWEN Hong，AN Yang，ZHANG Jun，XU Hui，LU Dao-min，CAO Yue-peng，NING Qiao-yi，WANG Ying【ABSTRACT】Objective：To study the effect of Usnea extract on apoptosis related factors in synovial cells of collagen induced arthritis（CIA）rats，and further explore its effect on joint inflammation and high mobility group protein B1（HMGB1）mediated cell autophagy in CIA rats.Methods：Fifty-four female Wistar rats were randomly divided into a blank control group，a model control group，a hydroxychloroquine group，and low，medium，and high dose groups of Usnea extract，with 9 rats in each group.The low，medium，and high dose groups of Usnea extract were administered with doses of 2 mg·kg-1，6 mg·kg-1，54 mg·kg-1，respectively，while the hydroxychloroquine group was administered with dose of 36 mg·kg-1.After establishing a CIA rat model，the drug was continuously administered by gavage for 28 d.ELISA method was used for detecting the concentration of HMGB1，IL-1β and TNF-α;RT-PCR was used to detect the expression of HMGB1，Beclin1 and PI3K C3 mRNA;and WB method was used to detect the the protein expression levels of HMGB1，Beclin1，and PI3K C3.Results：Compared with the blank control group，the content of HMGB1，TNF- α and IL-1β in each model group increased，the protein expression of HMGB1 and PI3K C3 increased，while the protein expression of Beclin1 decreased，with a statistically significant difference（P <0.05）.Compared with the model control group，the content of HMGB1，TNF-α and IL-1β in the middle and high dose groups of Usnea extract and the hydroxychloroquine group decreased，the content decreased，and the protein expression of HMGB1 and PI3K C3 decreased，while the protein expression of Beclin1 increased，with a statistically significant difference（P < 0.05）.Conclusion：Usnea extract up-regulates Beclin1 protein，down-regulates the expression of HMGB1 and PI3K C3related proteins，regulates cell autophagy，and reduces the concentration of HMGB1，IL-1β and TNF-α in serum of CIA rats，and reduces joint inflammation.【Keywords】 rheumatoid arthritis;Usnea extract;collagen induced arthritis;HMGB1;joint inflammation;autophagy;rats类风湿关节炎（rheumatoid arthritis，RA）是一种以滑膜炎为主要病理改变的自身免疫性疾病。

S1PR1-mediated IFNAR1 degradation modulates plasmacytoiddendritic cell interferon-α autoamplification由S1PR1介导的IFNAR1降解可以调节浆细胞样树突状细胞α-干扰素的自动扩增／信号放大摘要：Blunting immunopathology without abolishing host defense is the foundation for safe and effective modulation of infectious and autoimmune diseases.没有废除宿主防御机制的免疫病理钝化是安全、有效调节传染病和自身免疫性疾病的基础。

Sphingosine 1-phosphate receptor 1 (S1PR1) agonists are effective in treating infectious and multiple autoimmune pathologies; however, mechanisms underlying their clinical efficacy are yet to be fully elucidated.1-磷酸-鞘氨醇受体1（S1PR1）促效药对于治疗传染病和多种自身免疫性疾病是有效的，然而，其临床疗效的具体机制尚未被完全阐明。

Here, we uncover an unexpected mechanism of convergence between S1PR1 and interferon alpha receptor 1 (IFNAR1) signaling pathways.在本研究中，我们意外发现S1PR1与α-干扰素受体1（IFNAR1）信号通路之间的趋同／聚集机制。

Activation of S1PR1 signaling by pharmacological tools or endogenous ligand sphingosine-1 phosphate (S1P) inhibits type 1 IFN responses that exacerbate numerous pathogenic conditions.通过药理作用或内源性配体1-磷酸-鞘氨醇（S1P）发出信号激活S1PR1可以抑制1型干扰素应答，这将提供大量致病条件。

Friedenstein was the first person to identify multi-potential stromal precursor cells; he described the isolation, from the bone marrow, of spindle-shaped, clonogenic cells in monolayer cultures, which he defined as colony-forming unit fibroblasts (CFU-Fs). CFU-F-derived stromal cells can serve as feeder layers for the culture of haematopoietic stem cells (HSCs) and they can differentiate into adipocytes, chondrocytes and osteocytes both in vitro and after transfer in vivo 1. Friedenstein’s original observation was the basis of further studies showing that bone-marrow-derived stromal cells are the common predecessors of mesenchy-mal tissues . Recently, several studies have reported that multipotential stromal precursor cells can also differ-entiate into cells from unrelated germline lineages (in a process known as transdifferentiation )2,3 (BOX 1; FIG. 1). As a result of their supposed capacity for self-renewal and differentiation, bone-marrow-derived stromal cells were first considered as stem cells by Caplan and named mesenchymal stem cells (MSCs)4. However, in the bone marrow, stromal cells are a rare and heterogeneous population of cells that contain a mixture of progenitors at different stages of commitment to the mesodermal lineage and only a very small number of multipotential self-renewing stem cells, which have recently been identified as sub-endothelial cells that express CD146 (also known as MCAM)5. It is now accepted that most bone-marrow-derived progenitor stromal cells can be considered, after in vitro proliferation, to be MSCs 6. In addition to their potential for tissue repair, MSCs have been shown recently to have potent anti-proliferativeand anti-inflammatory effects. In this Review, we dis-cuss the functional features of bone-marrow-derived MSCs, describe their mechanisms of action and elu-cidate how these findings could be translated to the clinical setting.Definition of mesenchymal stem cellsMSCs, which can alternatively be defined as multipotent mesenchymal stromal cells, are a heterogeneous popula-tion of cells that proliferate in vitro as plastic-adherent cells, have fibroblast-like morphology, form colonies in vitro and can differentiate into bone, cartilage and fat cells 6 (FIG. 1). Although stromal cells that apparently fulfill these criteria for an MSC have been isolated from almost every type of connective tissue 7, MSCs have mainly been characterized after isolation from the bone marrow. Therefore, in this Review, we focus on bone-marrow-derived MSCs.MSCs that are cultured in vitro lack specific and unique markers. There is a general consensus that human MSCs do not express the haematopoietic markers CD45, CD34 and CD14 or the co-stimulatory molecules CD80, CD86 and CD40, whereas they do express variable levels of CD105 (also known as endoglin), CD73 (ecto-5′-nucleotidase), CD44, CD90 (THY1), CD71 (transferrin receptor), the ganglioside GD2 and CD271 (low-affinity nerve growth factor receptor), and they are recognized by the monoclonal antibody STRO-1. The variable expression level of these markers that has been observed probably arises from species differences, tissue source and culture conditions.*Department of Neurosciences,Ophthalmology and Genetics, University of Genoa, Italy. ‡Centre of Excellence for Biomedical Research, University of Genoa, Italy. §Advanced Biotechnology Center (ABC), Genoa, Italy. ||IRCSS Giannina Gaslini, Genoa, Italy. ¶Department of Experimental Medicine (DIMES), University of Genoa, Italy.Correspondence to A.U. e‑mail:auccelli@neurologia.unige.it doi:10.1038/nri2395Published online 18 August 2008Stromal cellsCells of non-lymphoid origin that form the framework of each organ. By expressing various molecules, these cells can support the adhesion, proliferation and survival of distinct cell subsets.Mesenchymal stem cells in health and diseaseAntonio Uccelli*‡§, Lorenzo Moretta ||‡¶ and Vito Pistoia ||Abstract | Mesenchymal stem cells (MSCs) are a heterogeneous subset of stromal stem cells that can be isolated from many adult tissues. They can differentiate into cells of the mesodermal lineage, such as adipocytes, osteocytes and chondrocytes, as well as cells of other embryonic lineages. MSCs can interact with cells of both the innate and adaptive immune systems, leading to the modulation of several effector functions. After in vivo administration, MSCs induce peripheral tolerance and migrate to injured tissues, where they can inhibit the release of pro-inflammatory cytokines and promote the survival of damaged cells. This Review discusses the targets and mechanisms of MSC-mediated immunomodulation and the possible translation of MSCs to new therapeutic approaches.Mesenchymal tissues These are embryonic tissues of mesodermal origin, consisting of loosely packed, unspecialized cells set in a gelatinous ground substance, from which connective tissue, bone, cartilage and the circulatory and lymphatic systems develop. T ransdifferentiationThe ability of a non-stem cell to transform into a different type of cell lineage, or when an already partially differentiated stem cell transforms into a different cell lineage or type. Stem cellsA subset of cells that has a self-renewing capacity andunder appropriate conditions can give rise to several mature cell lineages.Mesodermal lineageIn animals with three tissue layers, the mesoderm is the middle layer of tissue, between the ectoderm and the endoderm. In vertebrates, the mesoderm forms the skeleton, muscles, heart, spleen and many other internal organs.The role of MSCs in physiologyFormation of HSC niches in the bone marrow. Aftertransplantation into the bone marrow of non-obesediabetic–severe combined immunodeficiency (NOD–SCID) mice, MSCs have been shown to differentiate intopericytes, myofibroblasts, bone-marrow stromal cells,osteocytes, osteoblasts and endothelial cells, all of whichconstitute the functional components of the HSC niche thatsupport haematopoiesis8. The developing haematopoieticcells are retained in a quiescent state in the bone marrowuntil, after the appropriate stimulation, they differentiateand are then released in the sinusoidal vascular system.In the bone marrow, the niche stromal cells surroundthe HSCs and their progeny9(FIG. 2). Two types of nichehave been described in rodents. The ‘endosteal niche’ isformed by osteoblasts that line the endosteal surface ofthe trabecular bone, and the ‘vascular niche’ is composedof endothelial cells and CD146+ sub-endothelial stromalcells that lie at the abluminal side of bone-marrowsinusoids5. Stromal cells in both types of niche providea sheltering microenvironment that supports the main-tenance and self-renewal of HSCs by shielding themfrom differentiation and apoptotic stimuli that wouldotherwise challenge stem-cell reserves. Moreover, theniche also controls the proliferation and differentiation ofHSCs and the release of mature progeny into the vascularsystem. The regulation of HSC quiescence, through themaintenance of HSCs in the G0 phase of the cell cycle inthe endosteal niche, and the control of HSC proliferation,differentiation and recruitment in the vascular niche canbe ascribed to bone-marrow stromal cells10,11(FIG. 2).Anti-proliferative activity. Stromal-cell progenitors canpreserve the HSC pool in the bone marrow by maintain-ing HSCs in a quiescent state; in addition, terminallydifferentiated stromal cells such as fibroblasts and chon-drocytes seem to share anti-proliferative effects withtheir predecessors, as shown by their ability to inhibitT-cell proliferation12,13. Fibroblast-mediated modulationof T-cell responses is triggered by interferon-γ (IFNγ)14,which indicates that stromal cells in connective tissuesmight be involved in the homeostasis of peripheral leuko-cyte populations15. These results support the hypothesisthat stromal cells, at all stages of maturation, have anti-proliferative features that are in common with physiologi-cal stromal-cell niches, including the HSC niche.The effects of MSCs on immune cellsThe immunomodulatory effect of MSCs has only beendescribed recently, following the observation thatbone-marrow-derived MSCs suppressed T-cell pro-liferation16,17. These studies redirected the attention ofscientists away from the multipotentiality of MSCs towardstheir possible regulatory effects on immune cells, andthey paved the way for the characterization of the broadimmunoregulatory activities of MSCs.ture Reviews |ImmunologyMesoderm Figure 1 | The multipotentiality of MSCs. This figure shows the ability of mesenchymal stem cells (MSCs) in the bone-marrow cavity to self-renew (curved arrow) and to differentiate (straight, solid arrows) towards the mesodermal lineage. The reported ability to transdifferentiate into cells of other lineages (ectoderm and endoderm) is shown by dashed arrows, as transdifferentiation is controversial in vivo.HSC nicheThe microenvironment inside the trabecular bone cavity, which is composed of a specialized population of cells that has an essential role in regulating the self-renewal and differentiation of haematopoietic stem cells (HSCs). HaematopoiesisThe commitment and differentiation processes that lead from a haematopoietic stem cell to the production of mature cells of all blood lineages — erythrocytes, myeloid cells (macrophages, mast cells, neutrophils and eosinophils), B and T cells, and natural killer cells. SinusoidsBlood-filled spaces that lack the anatomy of a capillary. Sinusoids generally contain slow-flowing blood, which facilitates cellular interactions. Such vessels are found in the bone marrow and in the liver.Innate immunity. Myeloid dendritic cells (DCs) have afundamental role in antigen presentation to naive T cellsfollowing DC maturation, which can be induced by pro-inflammatory cytokines and/or pathogen-associatedmolecules. During maturation, immature DCs acquirethe expression of co-stimulatory molecules and upregu-late expression of MHC class I and class II moleculestogether with other cell-surface markers (such as CD11cand CD83). MSCs have been shown to inhibit thematuration of monocytes, and cord-blood and CD34+haematopoietic progenitor cells into DCs in vitro18–21(FIG. 3). Furthermore, mature DCs incubated withMSCs have decreased cell-surface expression of MHCclass II molecules, CD11c, CD83 and co-stimulatorymolecules, as well as decreased interleukin-12 (Il-12)production, thereby impairing the antigen-presentingfunction of the DCs18,20,22–24. MSCs can also decreasethe pro-inflammatory potential of DCs by inhibitingtheir production of tumour-necrosis factor (TNF)22.Furthermore, plasmacytoid DCs (pDCs), which arespecialized cells for the production of high levels oftype I IFN in response to microbial stimuli, upregulateproduction of the anti-inflammatory cytokine Il-10after incubation with MSCs22. Therefore, it is tempt-ing to speculate that the combined effects of MSCs onDCs and pDCs, as described in vitro, could translateinto potent anti-inflammatory and immunoregulatoryeffects in vivo.Natural killer (NK) cells are important effector cellsof innate immunity and they have a key role in anti v iraland anti-tumour immune responses owing to theircytolytic activity and production of pro-inflammatorycytokines25. The function of NK cells is tightly regu-lated by cell-surface receptors that transduce eitherinhibitory or activating signals. NK-cell-mediated lysisof target cells requires the expression of ligand(s) by thetarget cell that are recognized by activating NK-cellreceptors, together with low-level to absent expressionof MHC class I molecules by the target cell, which arerecognized by the MHC-class-I-specific inhibitory recep-tors of NK cells26,27. MSCs can inhibit the cytotoxic activityof resting NK cells by downregulating expression ofNKp30 and natural-killer group 2, member D (NKG2D),which are activating receptors that are involved in NK-cellactivation and target-cell killing28(FIG. 3).Freshly isolated, resting NK cells proliferate andacquire strong cytotoxic activity after culture with Il-2or Il-15. However, when resting NK cells are incubatedwith these cytokines in the presence of MSCs, NK-cellproliferation and IFNγ production are almost completelyabrogated28,29. Similar to resting NK cells, pre-activatedNK cells had decreased proliferation, IFNγ productionand cytotoxicity after culture with MSCs in vitro22,28–32.However, when the susceptibility of NK cells to MSC-mediated inhibition of proliferation was compared, pre-activated NK cells were found to be more resistant to theeffects of MSCs than were resting NK cells28.Conversely, both autologous and allogeneic MSCshave been shown to be killed by cytokine-activated, butnot resting, NK cells in vitro28,30,31(FIG. 3). The suscepti-bility of human MSCs to NK-cell-mediated cytotoxicitydepends on the low level of cell-surface expression ofMHC class I molecules by MSCs and the expressionof several ligands that are recognized by activatingNK-cell receptors29. Incubation of MSCs with IFNγpartially protected them from NK-cell-mediated cyto-toxicity through the upregulation of expression of MHCclass I molecules by MSCs28.Together, these findings support the possibility that,following encounter with MSCs in vivo, activated NKcells could undergo limited functional inhibition thatdoes not compromise their ability to kill MSCs. AsIFNγ protects MSCs from NK-cell-mediated lysis28, amicroenvironment rich in IFNγ might favour the inhi-bition of NK-cell function by MSCs, whereas in theabsence of IFNγ, the balance would be tilted towards theelimination of MSCs by activated NK cells. However,the in vivo relevance of these interactions might belimited only to cases of MSC transplantation.Neutrophils are another important cell type of innateimmunity that, in the course of bacterial infections, arerapidly mobilized and activated to kill microorganisms.After binding to bacterial products, neutrophils undergoa process known as the respiratory burst. MSCs have beenshown to dampen the respiratory burst and to delay thespontaneous apoptosis of resting and activated neutro-phils through an Il-6-dependent mechanism33(FIG. 3).previous studies have established a link between thedownregulation of the respiratory burst and an increaseture Reviews |ImmunologyFigure 2 | Stromal cells in the haematopoietic-stem-cell niche. In the bone marrow of trabecular bones, multipotential stromal progenitor cells at different stages of maturation contribute to the formation of the haematopoietic stem cell (HSC) niche. In the endosteal niche, stromal progenitor cells, together with osteoblasts, contribute to the maintenance of HSCs in a quiescent state (G0 phase of the cell cycle). Around sinusoids, angiopoietin-1 (ANG1)+CXC-chemokine ligand 12 (CXCL12)+CD146+ sub-endothelial stromal cells, perivascular stromal cells and sinusoidal endothelial cells also regulate HSC maintenance and control HSC proliferation, differentiation and recruitment to the vascular niche. The endosteal niche also contains self-renewing (dividing) HSCs and mobilized HSCs that are recruited to the vascular niche following proper activation.Respiratory burstA large increase in oxygen consumption and thegeneration of reactive oxygen species that accompanies the exposure of neutrophils to microorganisms and/or inflammatory mediators.Activation-induced cell deathA process by which activated, T -cell-receptor-restimulated T cells undergo cell death after engagement of cell-death receptors, such as CD95 or the tumour-necrosis factor receptor, or after exposure to reactive oxygen species.in the life span of neutrophils 34. MSC-mediated pres-ervation of resting neutrophils might be important in those anatomical sites where large numbers of mature and functional neutrophils are stored, such as the bone marrow and lungs 35.Adaptive immunity. After T-cell receptor (TCR) engagement, T cells proliferate and exert several effector functions, including cytokine release and, in the case of CD8+ T cells, cytotoxicity. The proliferation of T cells stimulated with polyclonal mitogens, allogeneic cells or specific antigen is inhibited by MSCs 16,17,22,36–45 (FIG. 3). This inhibition is not MHC restricted as it can be medi-ated by both autologous and allogeneic MSCs. MSC-mediated inhibition of T-cell proliferation depends on the arrest of T cells in the G0/G1 phase of the cell cycle 41,45. MSCs do not promote T-cell apoptosis, but instead support the survival of T cells that are subjected to overstimulation through the TCR and are committed to undergo CD95–CD95-ligand-dependent activation-induced cell death 45. The MSC-mediated anti-proliferative effect on T cells is associated with the survival of T cells in a state of quiescence that can be partially reversed by Il-2 stimulation 38.Inhibition of T-cell proliferation by MSCs has beenreported to lead to decreased IFN γ production both in vitro 22 and in vivo 38 and to increased Il-4 production by T helper 2 (T H 2) cells, which indicates a shift in T cells from a pro-inflammatory (IFN γ-producing) state to an anti-inflammatory (Il-4-producing) state 22.An important T-cell effector function is the MHC-restricted killing of virus-infected or allogeneic cells, which is mediated mainly by CD8+ cytotoxic T lympho-cytes (CTls). MSCs have been shown to downregulate CTl-mediated cytotoxicity 46 (FIG. 3). Human MSCs pulsed with viral peptides or transfected with mRNA from tumour cells were protected from lysis by CTls in vitro . pre-treatment with IFN γ increased the cell-surfaceture Reviews | ImmunologyFigure 3 | Possible mechanisms of the interactions between MSCs and cells of the innate and adaptiveimmune systems. a | Mesenchymal stem cells (MSCs) can inhibit the proliferation and cytotoxicity of resting naturalkiller (NK) cells and their cytokine production in vitro . These effects are mediated by prostaglandin E 2(PGE 2), indoleamine 2,3-dioxygenase (IDO) and soluble HLA-G5 (sHLA-G5) released by MSCs. Killing of MSCs bycytokine-activated NK cells involves the engagement of NKG2D (natural-killer group 2, member D) expressed by NK cells with its ligands ULBP3 (UL16-binding protein 3) or MICA (MHC class I polypeptide-related sequence A) expressed by MSCs, and of NK-cell-associated DNAM1 (DNAX accessory molecule 1) with MSC-associated PVR (poliovirus receptor) or nectin-2. b | MSCs inhibit the differentiation of monocytes to immature myeloid dendritic cells (DCs), skew mature DCs to an immature DC state, inhibit tumour-necrosis factor (TNF) production by DCs and increase interleukin-10 (IL-10) production by plasmacytoid DCs (pDCs). MSC-derived PGE 2 is involved in all of these effects. The immature DCs are susceptible to killing by cytokine-activated NK cells. The effect of MSCs on DCs impairs the stimulatory effect of mature DCs on resting NK cells and compromises antigen presentation to T cells, which cannot then undergo proliferation and clonal expansion. Finally, MSCs dampen the respiratory burst and delay thespontaneous apoptosis of neutrophils by constitutively releasing IL-6. c | Direct inhibition of CD4+ T-cell function depends on the release by MSCs of several soluble molecules, including PGE 2, IDO, transforming growth factor-β1 (TGF β1), hepatocyte growth factor (HGF), inducible nitric-oxide synthase (iNOS) and haem oxygenase-1 (HO1).Defective CD4+ T-cell activation impairs helper function for B-cell proliferation and antibody production. The inhibition of CD8+ T-cell cytotoxicity and of the differentiation of regulatory T cells mediated directly by MSCs are related to the release of sHLA-G5 by MSCs. In addition, the upregulation of IL-10 production by pDCs results in the increased generation of regulatory T cells through an indirect mechanism. MSC-driven inhibition of B-cell function seems to depend on soluble factors and cell –cell contact, but little is known about the identity of the molecules involved.HLA-GA non-classical MHC class Ib molecule that is involved in the establishment of immune tolerance at the maternal–fetal interface, the major soluble isoforms of which are HLA-G1 and HLA-G5.Notch signallingA signalling system comprising highly conserved transmembrane receptors that regulate cell-fate choice in the development of many cell lineages, and so are crucial for the regulation of embryonic differentiation and development.Antibody-secreting cellsA term that denotes both proliferating plasmablasts and non-proliferating plasma cells. The term is used when both cell types might be present. Multiple sclerosisA chronic inflammatory and demyelinating disease of the central nervous system. Multiple sclerosis involves an autoimmune response against components of myelin, which is thought to contribute to disease pathogenesis.expression of MHC class I molecules by MSCs but wasineffective at restoring CTl-mediated killing47,48, whichindicates that although MSCs inhibit CTl activity theyare not CTl targets.Regulatory T cells are a specialized subpopula-tion of T cells that suppress activation of the immunesystem and thereby help to maintain homeostasis andtolerance to self antigens. MSCs have been reportedto induce the production of Il-10 by pDCs, which, inturn, triggered the generation of regulatory T cells22,24.In addition, after co-culture with antigen-specific T cells,MSCs can directly induce the proliferation of regulatoryT cells through release of the immunomodulatory HLA-Gisoform HlA-G5 (ReF. 32)(FIG. 3).Taken together, these findings indicate that MSCscan modulate the intensity of an immune responseby inhibiting antigen-specific T-cell proliferationand cytotoxicity and promoting the generation ofregulatory T cells. In principle, from a clinical per-spective, excessive inhibition of T-cell responses byMSCs would render the host vulnerable to infectiousagents. However, fail-safe mechanisms might exist; forexample, MSCs express functional Toll-like receptors(TlRs) that, after inter a ction with pathogen-associatedligands, induce the proliferation, differentiation andmigration of MSCs and their secretion of chemokinesand cytokines49–51, and it has been shown that MSCslose the ability to inhibit T-cell proliferation due toimpaired Notch signalling after triggering of TlR3 andTlR4(ReF. 52). Therefore, it is possible that pathogen-associated molecules might reverse the suppressiveeffects of MSCs on T cells, thereby restoring efficientT-cell responses to pathogens52, but it is also possiblethat tissue stromal cells can instruct local immuneresponses after pathogen infections53.The second main cell type involved in adaptiveimmune responses is b cells, which are specialized forantibody production. Studies of the interactions betweenMSCs and b cells have produced different results, possi-bly as a result of the experimental conditions used41,54–57.Most published works to date indicate that MSCs inhibitb-cell proliferation in vitro41,54,56(FIG. 3). MSCs can alsoinhibit b-cell differentiation and the constitutive expres-sion of chemokine receptors56. These effects seem todepend on the release of soluble factors56 and on cell–cellcontact, possibly mediated by interactions between pro-grammed cell death 1 (pD1) and its ligands54. However,other in vitro studies have shown that MSCs couldsupport the survival, proliferation and differentiationto antibody-secreting cells of b cells from normal individ-uals57,58 and from paediatric patients with systemic lupuserythematosus57. Regardless of the controversial in vitroeffects, it should be emphasized that b-cell responses aremainly T-cell dependent and therefore the final outcomeof the interaction between MSCs and b cells in vivomight be significantly influenced by the MSC-mediatedinhibition of T-cell functions. Such an assumption issupported by the results of a study of experimental auto-immune encephalomyelitis (eAe) in mice injected witha proteolipid protein (plp) peptide, which is a modelof multiple sclerosis. In this model, the production ofantigen-specific antibodies in vivo was inhibited bythe infusion of MSCs, in addition to a significantdown r egulation of plp-specific T-cell responses, whichindicates that the two events were closely linked59.paradoxically, despite their broad immunosuppressiveactivities, it is possible that MSCs could function asnon-professional antigen-presenting cells (ApCs). lowconcentrations of IFNγ upregulate the expression of MHCclass II molecules by MSCs, which indicates that they couldact as ApCs early in an immune response when the levelsof IFNγ are low60,61. However, this upregulation of MHCexpression by MSCs, together with the ApC function, wasprogressively lost as IFNγ concentrations increased. Sucha mechanism could allow MSCs to function as conditionalApCs in the early phase of an immune response and laterswitch their function to immunosuppression60.Most of the immunomodulatory activities of MSCsdescribed here have been documented by in vitro experi-ments. As MSCs are derived from stromal progenitorcells that reside in the bone marrow, their potentialrole in the control of physiological immune responsesis unknown, despite the fact that the bone marrowmight be a site for the induction of T-cell responsesto blood-borne antigens62. However, it is possible thatMSC-mediated modulation of immune responses couldoccur in vivo following the infusion of in vitro-culturedMSCs after transplantation.If this hypothesis is correct, then infused MSCscould interfere with the interactions between DCs andNK cells. Mature DCs stimulate the proliferation andcyto t ocixity of NK cells and their cytokine production,whereas immature DCs are killed by NK cells25. The dualimmuno s uppressive effects of MSCs on DCs and restingNK cells could result in the accumulation of immatureDCs in vivo that are potentially amenable to NK-cell-mediated elimination, but also in the inhibition of NK-cellproliferation, cytotoxicity and cytokine production(FIG. 3). However, as discussed earlier, activated NK cellscan kill MSCs. Therefore, the functional outcome in vivowould be determined by the cytokine microenvironmentin which tripartite NK-cell–DC–MSC inter a ctions takeplace. In the absence of IFNγ, activated NK cells couldkill both immature DCs and MSCs. by contrast, in anIFNγ-enriched milieu, MSC-mediated inhibition ofimmune cells would prevail and target both DCs andNK cells. Such interactions between MSCs and immunecells might occur in vivo after MSC transplantation, butit should also be emphasized that the modulation of DCdifferentiation and function by tissue stromal cells couldbe viewed as an important mechanism that regulates alocal immune response53.As DCs are the main ApCs for T-cell responses,MSC-mediated suppression of DC maturation wouldpreclude efficient antigen presentation to and thereforethe clonal expansion of T cells (FIG. 3). Direct interactionsof MSCs with T cells in vivo could lead to the arrest ofT-cell proliferation, inhibition of CTl-mediated cyto-toxicity and generation of CD4+ regulatory T cells. Asa consequence, impaired CD4+ T-cell activation wouldtranslate into defective T-cell help for b-cell proliferationand differentiation to antibody-secreting cells. These。

抗人血管内皮生长因子嵌合抗体在真核细胞中的高效表达冉宇靓;杨治华;孙立新;遇珑;刘军;董志伟【期刊名称】《癌症（英文版）》【年(卷),期】2001(020)003【摘要】Objective：This study was designed to express chimeric anti-VEGF (vascular endothelial growth factor) antibody in dihydrofolate reductase-deficient Chinese hamster ovary (CHO-dhfr-)cells at high-level, and explore an optimum method to obtain high-level expression cells clone. Methods：The light chain and heavy chain genes of chimeric anti-VEGF antibody were induced into CHO-dhfr-cells using a novel eukaryotic high-level expression vectors system for genetic engineering antibodies. High-level expression was achieved after subcloning and several rounds of co-amplification of methotrexate (MTX). Biological features and productive amount of chimeric antibody was charactered by ELISA. Result：The cells strain that secret anti-VEGF chimeric antibody at the highest level of 28 μ g/ml was established. The cells were subcloned following each round of co-amplification of MTX, while greatly different results were obtained using three methods. The chimeric antibody contained constant regions of human immunoglobin and had the specificity against VEGF by ELISA. Conclusion：The anti-VEGF mouse-human chimeric antibody was expressed at high-level successfully in CHO cells. This may be an optimum method to obtain high-level expression cells clone for the eukaryotic high-level expression vectors system.%目的：在中国仓鼠卵巢（Chinese hamsterovary,CHO）细胞中高效表达有活性的抗人血管内皮生长因子（vascular endothelial growth factor,VEGF）嵌合抗体，并探索获得最佳表达的途径。