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◇基础研究◇摘要目的：观察常春藤皂苷元（hederagenin ，HDG ）改善银屑病小鼠皮肤损伤和炎症的作用与机制。
方法：通过在C57小鼠背部祛毛并连续涂抹咪喹莫特7d 建立小鼠银屑病动物模型，造模后1h 给予HDG 灌胃治疗。
总计设置正常组、模型组、模型+HDG 低剂量（25mg ·kg -1·d -1）、模型+HDG 高剂量（50mg ·kg -1·d -1）和模型+卤米松阳性对照组（每组8只小鼠）。
给药7d 后，对患处皮肤进行病理检测，以及炎症指标进行ELISA 、实时定量PCR 检测，Mincle 及其下游信号进行免疫组织化学、免疫荧光和Western blot 检测。
结果：与模型组比较，HDG 干预组皮肤病理损伤以及炎性细胞浸润均得到不同程度改善；实时定量PCR 和皮肤组织悬液ELISA 结果证实HDG 干预后小鼠皮肤中炎症因子IL-1β、IL-6和TNF-α的mRNA 及蛋白水平均比模型组降低（P <0.01），说明HDG 具有显著抗炎症作用；免疫组织化学和Western blot 结果表明，与正常组相比，模型组小鼠皮肤中Min-cle 的蛋白表达量显著增加（P <0.01），给予HDG 干预后明显下调（P <0.01）；免疫荧光证实模型组皮肤中Mincle 表达与巨噬细胞标志物F4/80共定位；Western blot 实验发现，HDG 在治疗组中不仅下调了Mincle 的蛋白表达，同时也下调了Mincle 下游信号Syk 和NF-κB 的蛋白磷酸化水平。
结论：HDG 可显著改善银屑病小鼠皮肤损伤和巨噬细胞相关炎症，其潜在分子机制可能与下调Min-cle/Syk/NF-κB 信号途径相关。
关键词常春藤皂苷元；Mincle ；皮肤损伤；炎症；银屑病中图分类号：R965.2文献标志码：A文章编号：1009-2501(2023)12-1339-08doi ：10.12092/j.issn.1009-2501.2023.12.003银屑病是一种慢性丘疹鳞状皮肤病，其主要特点是遗传性和复发性，还可能并发其他疾病，如心血管疾病、糖尿病和关节炎等［1-4］。
昆明医学院学报2012，（1B ）：31～34Journal of Kunming Medical UniversityCN 53-1049/R生物制剂在炎症性肠病治疗中的现状和前景吴汗1，2），杨太明2）（1）昆明医学院第一附属医院消化内科，云南昆明650031；2）云南省临沧市人民医院消化内科，云南临沧677000）［摘要］炎症性肠病（IBD ）是一种发生在肠道的病因不明的慢性非特异性炎性病变，包括溃疡性结肠炎（UC ）和克罗恩病（CD ），其发病原因尚不清楚，发病机制尚未完全阐明．目前，随着免疫学和生物学的发展，越来越多的生物学制剂用于IBD 的治疗，使炎症性肠病的治疗在传统治疗的基础上，增加了对疾病本身的并发症或治疗有关的并发症的预防，从而改善了患者的生活质量，减少了住院率和外科手术率．这也是生物药物治疗的发展方向．就IBD 的生物药物治疗现状和前景作一简要综述．［关键词］炎症性肠病；生物治疗；临床应用［中图分类号］R574.62［文献标识码］A ［文章编号］1003－4706（2012）1B －0031－04Cur r ent St at us and Pr ospect s of Biological Agent Ther apy forInflammat or y Bowel DiseaseWU Han 1，2），YANG Tai －ming 2）（1）Dept.of Gastroenterology ，The Affiliated Hospital of Kunming Medical University ，Yunnan Kunming 650031；2）Dept .of Gastroenterology ，The People ’s Hospital of Lincang ，Lincang Yunnan 677000，China ）［Abstract ］Inflammatory bowel disease （IBD ），is a chronic non-specific inflammatory disorder with unknown etiology ，including ulcerative colitis and Crohn ’s disease （CD ）．With the development of immunological and biological studies ，more and more biological agents have been studied and used for the treatment of IBD ，Consequently ，the clinical outcome of IBD is unsatisfying ．A variety of new biological therapies have being developed in recent years.These therapies are shown to be helpful in inducing remssion ，preventing complication ，inproving life quality of the patients ，and reducing hospitalization rates and surgical rates ．which is a direction for future development of agent therapy ．This review summarized the curret status and prospects of biological agent therapy for IBD.［Key words ］Inflammatory bowel disease ；Biological therapy ；Clinical application ［作者简介］吴汗（1985～），男，云南临沧市人，在读硕士研究生，主要从事消化内科临床工作.［通讯作者］杨太明．E-mail:lcytm@炎症性肠病（iinflammatory bowel disease ，IBD ）包括克罗恩病（Crohn ’s disease ，CD ）和溃疡性结肠炎（ulcerative colitis ，UC ），是一种发生在胃肠道的慢性炎症性疾病，其临床表现为腹痛、腹泻、便血、体重下降等，其特点是病情迁延不愈，反复发作，且发病原因尚未明确．近年来研究发现在IBD 患者的肠粘膜组织内有大量激活的T 细胞、B 细胞、NK 细胞、巨噬细胞、树突状细胞，这些细胞能分泌高水平的促炎症细胞因子，表达高水平的细胞因子受体、趋化因子受体、整合素等．而在肠粘膜组织内的毛细血管的内皮细胞则表达高水平的趋化因子，这些分子间的相互作用能诱导血液循环中的白细胞向组织内移动、归巢、浸润，从而使肠粘膜发生炎症损伤．IBD 治疗方案很多，其中IBD 的传统治疗药物主要包括氨基水杨酸类药物（5-氨基水杨酸、柳氮磺胺吡啶）、糖皮质激素、免疫第33卷昆明医学院学报32抑制剂硫唑嘌呤（AZA）、6-巯基嘌呤（6-MP）、环磷酰胺、环孢素A等．大多数患者经这些药物治疗后临床症状均可得到缓解，但仍有一部分患者的病情无法得到有效控制．近几年来，随着对IBD认识的不断深入，多项治疗IBD的大规模临床试验的开展，都证实了生物制剂对IBD的治疗有效，现已有多种制剂应用于临床．例如英芙利昔（infliximab）、阿达木单抗（Adalimamab）、赛妥珠单抗（Certolizumab pegol）、那他珠单抗（Natalizamab）等，其中以英芙利昔应用最为广泛．目前，随着生物制剂在临床上应用的不断增加，应用时间的不断延长，生物制剂的治疗效果也越来越受关注，本文就对治疗IBD的生物制剂的现状和前景作一概括．1促炎细胞因子抑制剂1.1肿瘤坏死因子（TNF）抑制剂近年来的研究发现，在UC患者血液、结肠粘膜组织、肠道灌洗液中，TNF-α明显增加，其中还发现大量活化的单核-巨噬细胞和T细胞．抗TNF-α抗体通过与TNF的特异性结合，促进粘附分子下调．同时可使表达TNF的炎细胞凋亡，从而使炎症消退．目前治疗IBD的抗TNF制剂主要有3种：英芙利昔（Infliximab）、阿达木（Adalimuma，D2E7）和聚乙二醇化西地丽珠（Certolizumab，CDP-870）．根据中国2007年IBD 治疗规范的共识意见，IFX是唯一被推荐使用的抗TNF-α单抗，其适应证仅是传统治疗无效或有肛周病变的CD[1]，但世界胃肠病组织2010年IBD诊疗指南中，3种抗TNF-α单抗均被推荐使用，而且适应证中除中、重度CD，难治性和有肛周病变的CD外，还包括重度或激素及免疫抑制剂无效或不能耐受的UC患者[2]．1.1.1英芙利昔（Infliximab）英芙利昔是最早用于治疗IBD的生物制剂，通过结合跨膜的TNF，抑制TNF细胞的表达功能，并通过Fc段介导T细胞的补体结合作用诱导细胞凋亡，从而产生抗体依赖的细胞毒作用[3]．英芙利昔最先用于CD治疗，后批准用于治疗UC[4]，IFX对IBD的诱导缓解，临床症状改善，内镜下粘膜愈合，瘘管修复等均有显疗效．常规使用剂量为5mg/kg，在第0、2、6周静脉滴注，然后每间隔8周静脉滴注1次，以防止复发．一般用药2周起效，每8周重复1次可持续1a，达到较好的临床缓解．Schnitzler等[5]在对英芙利昔单抗治疗CD的长期临床效果研究发现：在接受IFX治疗的614例患者中，10周后有547例产生应答，占89.1%，其后继续给予维持治疗，并在27～83个月进行随访，在长期治疗中，有347例患者有效，占其中的63.4%．在这些患者中，有68.3%一直接受英芙利昔单抗治疗，其中31.7%因一直处于缓解状态而停止了治疗，12.8%因不良反应而停药，21.6%失去应答．接受程序性治疗的患者，比按需用药的患者，其住院率、手术率和停用激素的比例均降低．这组5a随访的队列研究显示：通过长期英芙利昔单抗治疗，可以维持该药的远期效果．陈白莉等[6]应用英芙利昔单抗治疗10例难治性CD患者，30周后有临床缓解和粘膜愈合各有4例．对于UC治疗，由Rutgeerts等和Sandborn等进行的两个国际多中心、随机、安慰剂对照试验（ACT1和ACT2）报告了治疗的远期效果．通过54周随访，发现英芙利昔单抗组与安慰剂组UC患者的结肠清除率分别为10%和17%（P＝0.02），2组患者住院率分别为20%人/年和40%人/年（P＝0.003）．结果提示：IFX有较好的远期疗效[7]．李世荣[8]等对比观察了14例IBD患者的治疗效果，结果显示，UC患者的疗效好于CD 患者的疗效，主要原因在于UC患者多有便血表现，能够及早就医，而CD患者多出现合并症才引起重视，诊断较晚而影响疗效．根据美国FDA关于infliximab的报告，infliximab的适应症包括：难治性的克罗恩病、激素依赖性的克罗恩病、急性中重度的克罗恩病、慢性难治性的溃疡性结肠炎、IBD并发其他系统性疾病（如强制性脊柱炎、坏疽性脓皮病、慢性葡萄膜炎等）等[9]．不良反应有：（1）感染：应用IFX制剂可使IBD患者的机会性感染增加，如细菌感染、分枝杆菌感染、侵袭性真菌感染等，极少可发生严重感染，包括肺炎、感染中毒症、蜂窝织炎、皮肤和泌尿系感染等；（2）过敏反应：使用IFX的患者中，少数出现过敏反应；（3）自身抗体和药物性狼疮．长期使用IFX 的患者，血清抗核抗体（ANA）和抗双链DNA （ds-DNA）抗体的滴度增加，出现自身免疫反应；（4）淋巴瘤或恶性肿瘤：TNF-α对肿瘤生长有抑制作用，因此，使用IFX后对肿瘤的发生可能有一定促进作用；（5）心力衰竭：目前对于心功能Ⅲ、Ⅳ级患者不主张使用IFX．1.1.2阿达木单抗（Adalimumab）阿达木单抗通过结合膜表面的TNF，活化补体和发挥抗体介导补体依赖性的细胞毒作用．不仅可以诱导CD的临床缓解[10，11]，而且对应用英芙利昔单抗失败或不能耐受英芙利昔单抗的病例有一定疗效[12]．根据美国吴汗，等．生物制剂在炎症性肠病治疗中的现状和前景33第1B期FAD决议，其临床适应症包括：克罗恩病以及克罗恩病合并类风湿性关节炎、银屑病关节炎的治疗．禁忌症包括：活动性感染（如肺结核感染）、神经系统疾病、淋巴瘤等，不良反应包括：局部注射部位的反应、重度感染、神经功能的损害以及淋巴系统的影响（如淋巴瘤等）[13]．Oussalah等[14]对13例UC患者进行长期随访，在第1、3、6、23个月时缓解率分别为92.3%、84.6%、60.6%、32.0%，最终在13例患者中仅有6例患者行结肠切除术，这说明阿达木单抗的长期治疗，可以很大程度的减少手术率．Colombel等[15]对合并瘘管的CD患者应用阿达木单抗治疗的远期疗效进行观察，对研究的854例CD患者用英芙利昔单抗治疗，其中117例合并瘘管的患者，以1:1:1的比例随机分入每周阿达木单抗40mg组（40例）、每2周40mg组（30例）和安慰剂组（47例）．结果发现，阿达木单抗组每天伴有引流液的瘘管数目明显比安慰剂组少．对继续用阿达木单抗维持者，随访56周后发现瘘管愈合率90%．对于阿达木单抗的用法，美国胃肠病学会推荐的是皮下注射，0周160mg，第2周80mg，治疗有效者，每2周40 mg维持：4周无效者，不再使用[16]．1.1.2赛妥珠单抗（Cert olizumab，或CDP－870）赛妥珠单抗于2008年被美国FDA批准应用于临床，其适应症包括：中重度的CD患者．不良反应有：注射部位的局部反应、上呼吸道感染、泌尿系统感染以及关节疼痛[17]．个别病例报道可以导致致命的感染并发症（如真菌、结核菌、机会菌感染等）．该药的推荐使用剂量为0周，2周和4周皮下注射400mg，有效者每4周400mg维持治疗．1.2IFN-γ抑制物（Font olizumab）IFN-γ抑制物对治疗节段性肠炎和伴有高反应蛋白水平的患者有很好疗效．有试验对活动性CD患者静脉滴注4或10mg/kg，8周的临床有效率分别为69%和67%，安慰剂对照组为32%[18]．2选择性细胞粘附分子抑制剂该制剂主要是通过阻断活化的淋巴细胞和单核细胞从血管向组织移动，从而减轻CD患者的肠粘膜炎症反应，改善临床症状和组织学表现．临床上有那他珠单抗（Natalizumab）和人体化的α4β7整合素拮抗剂（MLN-02）．2.1那他珠单抗（Natalizumab）那他珠单抗是第一种新型的选择性细胞粘附分子抑制剂，通过对白细胞归巢和粘附活动的抑制，减少白细胞进入组织，达到减轻炎症反应的目的．应用方法为0，4和8周300mg静脉滴注，有应答者，每4周300mg静脉点滴．目前已被美国FDA 批准应用于临床．其临床适应症包括：顽固性的中重度克罗恩病．主要的不良反应包括：进行性多灶性白质脑病、严重肝损害、呼吸道感染、关节痛、头痛、输液反应等．2.2人体化的α4β7整合素拮抗剂（MLN－0002）该制剂是通过阻止白细胞与血管内皮粘附，从而促进炎症愈合．仅用于炎症性肠病．Feagan等应用M LN0002对181例UC患者进行多中心、双盲、安慰剂对照试验．用药为MLN00020.5mg/kg 组，2mg/kg组和安慰剂组，经过治疗6周后，缓解率分别为33%、32%和14%（P＝0.002）．内镜下缓解率为28%、12%和8%（P＝0.007）[19]．3其他生物学治疗药物随着分子生物学和临床免疫学的不断发展，研究发现有多种生长因子参与结肠粘膜的炎症免疫反应，与IBD的的发病和转归密切相关．因此，在国外，已经有多种生长因子制剂投入临床治疗，并取得一定疗效．3.1粒细胞集落刺激因子（granulocyte macrop-hage-colony stimulating factor，GM-CSF）粒细胞集落刺激因子是一种造血生长因子，能刺激肠道内在的自然免疫系统来抑制炎症反应，给药方式为皮下注射，剂量6ug/kg．目前临床适应症为：中重度的克罗恩病[20]．不良反应是：注射部位的局部反应以及骨关节痛．3.2表皮生长因子（epidermal growt h fact or，EGF）EGF能促进细胞生长、增殖和分化，是一种强效细胞保护因子，主要通过局部灌肠给药．其临床适应症为：轻中度溃疡性结肠炎．不良反应现今尚未清楚．3.3人源性抗CD3单克隆抗体visilizumab（HuM291）该制剂是一种无FcR片段的人源性抗CD3单克隆抗体，对CD3发挥作用，诱导活化的T细胞凋亡．4生物治疗的前景和展望随着人们对IBD发病机制的深入了解，以及免疫学和生物工程的参与，生物制剂的治疗给临床医学带来了新的前景，为IBD患者带来新的希望和曙光．分子生物制剂不仅可以治疗原因不明的炎性疾病，而且还能有效预防合并症，阻止疾病的进展．通过对有关肠粘膜免疫病理学的深入了解和各种临床试验的开展，以及各学科的不断成熟，将会研制出更多的显效迅速、使用安全、耐受性好、用药方便的生物制剂，给广大的IBD患者带来更多的治疗选择．［参考文献］［1］中华医学会消化病学分会炎症性肠病协作组.对我国炎症性肠病诊断治疗规范的共识意见［J］．中华消化杂志，2007，12（3）：488.［2］CHARLES N B，M ICHAEL F，KRABSHUIS J H，et al.W-World gastroenterology organization practice guidelines forthe diagnosis and management of IBD in2010［J］．In-flamm Bowel Dis，2010，16（5）:112.［3］VAN DEVENTER S J．Anti-tumour necrosis factor thera-py in Crohn’s disease:where are we now［J］．Gut，2002，51（4）：362－363.［4］YAM AM OTO-FURUSHO J K．Innovative therapeutics for inflammatory bowel disease［J］．World J Gastroenterol，2007，13（13）：1893－1896.［5］SCHN IT ZLER F，F ID DER H，FERRANCE M，et al．Long-term outcome of treatment with infliximab in614pa-tients with Chron’s disease results from a single centre co-hort［J］．Gut，2009，58（4）：492－500．［6］陈白莉，陈文湖，高翔，等．新型生物制剂英芙利昔治疗克罗恩病10例［J］．中华消化杂志，2008，28（12）：831－834．［7］SAND BORN W J，RUTGEERTSP，FEAGANBG，et al.C-olectomy rate comparison after treatment of ulcerative colitiswith place boor infliximab［J］．Gastroenterol，2009，137（4）：1250－1260.［8］李世荣，陆小娟，盛剑秋，等．溃疡性结肠炎和克罗恩病的英芙利昔治疗效果比较［J］．胃肠病学和肝病学杂志，2010，19（7）：38－41.［9］RUTGEERTS P，VAN ASSCHE G，VERM EIRE S．Revi-ew article：Infliximab therapy for inflammatory bowel dis-ease-seven years on［J］．Aliment Pharmacol Ther，2006，23（7）：451－463.［10］HANAUER S B，SA NDBORN W J，RUTGEERTS P，et al.Human anti-tumor necrosis factor monoclonal antibody（adalimumab）in Chron’s disease the CLASSIC-1trial［J］．Gastroenterol，2006，130（2）：323－333.［11］SANDBORN W J，HANAUER S R，RUTGEERTS P，et al.adalimumab for maintenance treatment of Crohn’s diseaseresults of the CLASSIC-Ⅱtrial［J］．Gut，2007，56（9）：1232－1237.［12］SANDBORN W J，HANAUER S B，LOFTUS EV J R，et al.An open-label study of the human anti-TNF monoclonalantibody adalimumab in subjects with prior loss of responseor in tolerance to infliximab for Crohn’s disease［J］．Am JGastroenterol，2004，99（10）：1984－1989.［13］HANAUER S B，SANDBORN W J，RUTGEERTS P，et al.Human anti-tumor necrosis factor monoclonal antibody（adalmumab）in Crohn’s disease:the CLASSIC-1trial［J］．Gastroenterology，2009，130（9）：323－591.［14］OUSSALAH A，LACLOTTE C，CHEVAUX JB，et al.Long-term outcome of adalimumab therapy for ulcerativecolitis with intolerance or lost response to infliximab:a sin-gle-centre experience［J］．Aliment Pharmacol Ther，2008，28（7）:966－972.［15］COLOM BEL J F，SCHW ARTZ D A，SANDBORN W J，et al．Adalimumab for the treatment of fistulas in patientswith Crohn’s disease［J］．Gut，2009，58（7）：940－948.［16］HANAUER S B，SANDBORN W J，RUTGEERTS P，et al.Human anti-tumor necrosis factor monoclonal antibody（adalmumab）in Crohn’s disease:the CLASSIC-1trial［J］．Gastroenterology，2006，130（6）：323－591.［17］SCHREIBER S，KHALIQ－KAREEM I M，LAWRANCE I C，et al．M aintenance therapy with certolizumab pegol forCrohn’s diaease［J］．N Engl J M ed，2007，357（8）:239－250.［18］HOM M ES D W，M IKHAJLOVA T L，STOINOV S，et al．Fontolizumab，ahumanized anti-interferon gamma anti-body，demonstrates safety and clinical activity in patientswith moderate to severe Crohn’s disease［J］．Gut，2008，55（8）：1131－1137.［19］FEAGAN B G，GREENBERG G R，W ILDG，et al．Treat-ment of active Crohn’s disease with M LN0002.A human-ized antibody to the alpha4beta7integrin［J］．Clin Gas-troenterol H epatol，2008，6（12）：1370－1371.［20］TAKAZOE M，M ATSUI T，M OTOVA S，et al．Sargramos-tim in patients with Crohn’s disease;result of a phase1-2study［J］．J Gastroenterol，2009，44（10）：535－543.（2012－01－06收稿）第33卷昆明医学院学报34。
慢病毒介导骨形态发生蛋白2和血管内皮生长因子165双基因转染促进骨髓间充质干细胞向成骨细胞分化周桢杰;李强;李诗鹏;陶旋;马跃刚【摘要】背景:骨形态发生蛋白2(bone morphogenetic protein 2,BMP-2)和血管内皮生长因子165(vascular endothelial growth factor 165,VEGF-165)均为骨修复过程中必不可少的细胞因子,利用慢病毒转染干细胞研究双因子作用下细胞的生物学改变具有重要意义.目的:探讨慢病毒介导人BMP-2和人VEGF-165双基因转染(2次转染)骨髓间充质干细胞的可行性以及转染后细胞的诱导成骨性能.方法:将骨髓间充质干细胞分为4组:①未转染组;②空载组:通过2次相同感染复数的空载慢病毒转染细胞;③BMP-2组:转染了单个目的基因的细胞(Lv-BMP-2/GFP);④BMP-2/VEGF-165组:通过2次转染后携带双目的基因的细胞(Lv-BMP-2/GFP,Lv-VEGH-165/RFP).转染后不同时间点Western Blot检测目的蛋白表达,MTT法检测各组细胞增殖活性,转染后14 d检测各组细胞碱性磷酸酶活性.结果与结论:①空载组、BMP-2组、BMP-2/VEGF-165组在荧光显微镜下均可见相应的绿色及红色荧光蛋白表达;②转染后3 d,BMP-2组、BMP-2/VEGF-165组均有相应目的蛋白高表达;转染后7,14,21 d,目的蛋白检测无明显变化(P>0.05);③BMP-2/VEGF-165组增殖稍快于BMP-2组(P<0.05),BMP-2组明显快于未转染组、空载组(P<0.05);④BMP-2组、BMP-2/VEGF-165组碱性磷酸酶活力明显高于未转染组、空载组;⑤结果表明,慢病毒介导的hBMP-2和hVEGF-165双基因经2次转染成功转入兔骨髓间充质干细胞内,并长期稳定表达,该双因子的表达能通过刺激细胞碱性磷酸酶生成促使细胞更好的向成骨细胞分化.【期刊名称】《中国组织工程研究》【年(卷),期】2018(022)025【总页数】6页(P3950-3955)【关键词】骨髓间充质干细胞;骨形态发生蛋白2;血管内皮生长因子165;慢病毒;基因转染;细胞增殖;碱性磷酸酶;干细胞;国家自然科学基金【作者】周桢杰;李强;李诗鹏;陶旋;马跃刚【作者单位】桂林医学院附属医院,广西壮族自治区桂林市 541001;桂林医学院附属医院,广西壮族自治区桂林市 541001;桂林医学院附属医院,广西壮族自治区桂林市 541001;桂林医学院附属医院,广西壮族自治区桂林市 541001;桂林医学院附属医院,广西壮族自治区桂林市 541001【正文语种】中文【中图分类】R394.2文章快速阅读：文题释义：慢病毒载体：是以人类免疫缺陷Ⅰ型病毒为基础发展起来的基因治疗载体，其对分裂细胞和非分裂细胞均具有较强的感染能力。
·19·维持基因组序列信息的完整性对于生物体的存在至关重要，细胞应对各种类型损伤因子所导致的DNA损伤主要是通过激活复杂而精细的DNA 损伤应答 （DNA damage response，DDR ） 通路［1］。
具有细胞毒性的双链断裂损伤 （double strand breaks，DSBs ） 是最为严重的DNA损伤形式之一，未得到正确处理的DSBs除可增加细胞的致死率外，还会增加· 论著 ·DNA损伤修复通路因子53BP1在骨髓干细胞自我更新和分化发育中的作用尤放，王美莲（中国医科大学基础医学院病原生物学教研室，沈阳 110122） 摘要 目的 探讨DNA 损伤修复通路因子53BP1在骨髓干细胞自我更新及定向分化过程中发挥的作用。
方法 构建53BP1全身敲除C57/6小鼠模型，免疫荧光染色检测53BP1在细胞中的定位，Western blotting 对53BP1蛋白表达水平进行检测及鉴定；流式细胞术进行细胞分选和计数、细胞周期检测、以及细胞表面分子marker 分析，对比WT 组和Mut 组细胞生长速率以及定向分化过程，Co -IP 检测分析表观遗传学磷酸化修饰蛋白，SPSS 22.0对数据进行统计学分析。
结果 体内和体外实验证实伴随KSL 细胞分化发育进行，53BP1表达含量显著增加，53BP1基因敲除可影响KSL 细胞的自我更新和定向分化以及表面分子标记的表达；53BP1基因敲除与否并不显著影响处于细胞周期G 1/G 0的KSL 细胞百分含量；53BP1可影响磷酸化修饰蛋白的表达水平，53BP1基因敲除后磷酸化修饰蛋白p -H4 （k20） 和p -H3 （k79） 表达水平明显增加，但p -ATM 和p -53-Ser -20含量显著减少。
结论 53BP1参与并影响骨髓干细胞的自我更新及定向分化过程，53BP1基因敲除可显著影响磷酸化蛋白p -H4 （k20）、p -H3 （k79）、p -ATM 和p -53-Ser -20的表达水平。
The Promise and Perils of Stem Cell TherapeuticsGeorge Q.Daley1,2,3,*1Stem Cell Transplantation Program,Division of Pediatric Hematology/Oncology,Manton Center for Orphan Disease Research,Howard Hughes Medical Institute,Children’s Hospital Boston and Dana Farber Cancer Institute;Division of Hematology,Brigham and Women’s Hospital;and Department of Biological Chemistry and Molecular Pharmacology,Harvard Medical School,Boston,MA02115,USA2Broad Institute,Cambridge,MA02142,USA3Harvard Stem Cell Institute;Boston,MA02138,USA*Correspondence:george.daley@DOI10.1016/j.stem.2012.05.010Stem cells are the seeds of tissue repair and regeneration and a promising source for novel therapies. However,apart from hematopoietic stem cell(HSC)transplantation,essentially all other stem cell treatments remain experimental.High hopes have inspired numerous clinical trials,but it has been difficult to obtain unequivocal evidence for robust clinical benefit.In recent years,unproven therapies have been widely prac-ticed outside the standard clinical trial network,threatening the cause of legitimate clinical investigation. Numerous challenges and technical barriers must be overcome before novel stem cell therapies can achieve meaningful clinical impact.Cell Therapeutics:The Current Standard of CareIn the twentieth century small molecule and protein drugs proved remarkably successful in restoring health and extending life span, but in the twenty-first century our aging population will face an increasing burden of organ failure and neurodegenerative disease.Such conditions are unlikely to be cured by drugs alone and instead call for restoration of tissue function through novel therapeutic approaches.Transplantation of whole organs—heart,lung,liver,kidney,small bowel,and pancreas—has become routine in modern medicine and has saved countless lives,while grafts of the skin and cornea for burns or ocular injury and transfusions of red blood cells and platelets for disease-related or chemotherapy-induced cytopenias are likewise widely employed tissue and cell therapies.However,current therapeutic strategies either are limited by donor availability and immunologic barriers or pertain to only a minor range of conditions.For the many diseases and disorders of aging for which there is no cure,innovative applications of tissue engineering and novel cell therapies derived from pluripotent and tissue-restricted stem cells represent major frontiers for the future. Hematopoietic stem cells(HSCs),the therapeutic constituents of whole bone marrow and umbilical cord blood,have been the most widely employed stem cell therapy.When successful,HSC transplantation can be curative for scores of genetic blood disorders like thalassemia and immune deficiency and for malig-nancies like leukemia and lymphoma.HSC transplantation is undoubtedly the most successful application of stem cells in medicine,yet for many conditions success rates remain frustrat-ingly low and morbidity and mortality unacceptably high.The need for precise molecular matching of donor and recipient means that many patients lack a suitable donor,either within their own family or in the public at large,even when databases list many millions of potential unrelated donors.When a match can be found,minor mismatches between donor and recipient frequently incite graft versus host disease(GVHD),an attack of the donor immune effector T cells against host tissues that results in skin rash,mucositis,diarrhea,and liver and lung destruction.GVHD is a major cause of treatment associated morbidity and mortality.Finally,grafts can fail,and disease can relapse.Although it is difficult to give a precisefigure for the over-all success rate for HSC transplantation,even an optimist would acknowledge that some50%of patients are left without a cure or with a permanent disability.Thus,even our most successful form of stem cell therapy remains a heroic effort,reserved only for the sickest patients who have no better alternative.Lessons from the Historical Development of HSC TransplantationThe evolution of HSC transplantation from its experimental origins to its acceptance as a standard of care in medicine is a tale that is both inspiring and cautionary.E.Donnall Thomas and colleagues were thefirst to perform marrow transplantation for otherwise fatal leukemia in the1950s(Thomas et al.,1957). The rationale was predicated upon the known capacity for radi-ation to suppress leukemic hematopoiesis and studies demon-strating that injections of marrow rescued mice from otherwise lethal radiation exposure(Jacobson et al.,1951;Lorenz et al., 1951).Thomas wrote in a memoir in2005,‘‘These patients inspired us to speculate that it might be possible to destroy leukemic cells and normal marrow by lethal whole body irradia-tion,with reconstitution of marrow by marrow transplantation.’’Arguably,thefirst studies in humans were founded upon rather minimal evidence of efficacy in rodent models,and Thomas further noted,‘‘We recognized that it would be important to do similar studies in an animal model.[and]decided to move forward with studies of man and dog at the same time’’(Thomas, 2005).Indeed,Thomas and colleagues suffered considerable failure in preclinical canine models and witnessed the deaths of many scores of patients,which prompted great skepticism about whether the human experiments should continue.Never-theless,Thomas and his intrepid team of investigators forged ahead.It took almost two decades before advances in research on tissue matching to define compatible donor-recipient pairs, and improved treatment of graft versus host disease and the infectious complications of marrow transplant allowed marrow transplantation to achieve consistent success in the late1970s.740Cell Stem Cell10,June14,2012ª2012Elsevier Inc.Some important principles emerge from this lesson in the history of HSC transplantation.First,the risk of the intervention should be commensurate with the severity of the underlying condition to be treated.The aggressively malignant nature of the conditions being treated—fatal leukemia and marrow apla-sia—meant that thefirst practitioners of marrow transplantation were justified and even compelled to attempt heroic and poten-tially highly toxic interventions for invariably fatal diseases. Second,although human biology is only partially predictable from animal models,preclinical animal models remain a key element in the scientific development of novel therapies.At the beginning of human marrow transplantation,it was understood that identical twins accepted skin and solid organ grafts,but only a minority of the time did siblings.Experiments in the murine and canine marrow transplantation models reflected similar transplantation barriers.Notwithstanding these sobering limita-tions,the early practice of marrow transplant in patients pro-ceeded despite a lack of robust evidence in animal models for graft acceptance between unrelated individuals.Only later were methods for lymphocyte matching developed(the ante-cedent to HLA typing),which was the key development in advancing the success of marrow transplantation.Finally,impor-tant and fundamental insights into therapeutic mechanisms wererequired before the eventual success of clinical translation of HSC transplantation therapies.With the benefit of hindsight,one could argue that the earliest human transplants were premature and doomed to fail.One might question whether a therapy as toxic as marrow transplant, with so little evidence for success in animal models prior to testing in humans,could emerge in the current era.Under today’s more rigorous regulatory climate,institutional review boards weigh risks and potential benefit on behalf of patients, insist on an impartial process of informed consent to minimize misconceptions about therapeutic potential,and monitor adverse events in the course of clinical trials.Indeed,one might reasonably conclude that today’s IRBs might not have approved the early studies of Thomas and colleagues,but if they had, would have interceded to stop the experiments when the high incidence of treatment-related mortality became apparent. The conjecture that modern-day IRBs might not approve the early experiments in HSC transplant does not imply that HSC transplant would not emerge under the current regulatory climate.On the contrary,I believe that bone marrow transplant could be developed within today’s environment of strict clinical research regulation,although by a more conservative path that would spare considerable patient morbidity and mortality.As we learned from premature attempts at gene therapy in the early 1990s,new therapeutic technologies require considerable understanding of fundamental mechanisms before they can be delivered with confidence.Indeed,roughly70%of early phase clinical trials of pharmaceuticals fail and over50%at phase III (Ledford,2011),and thus it stands to reason that significant resources are squandered because of the imprecision of early stage clinical research.Yet,especially with novel technologies, clinical experimentation proceeds energetically,because hope triumphs over experience.From this author’s perspective, a conservative approach to clinical translation of stem cell ther-apies is warranted at this time,not because stem cell treatments are excessively risky(though some may yet prove to be),but rather because our understanding of the mechanisms by which stem cells might prove useful,and in which diseases,remains primitive.In a climate where government and philanthropic funds for fundamental research are increasingly scarce,and invest-ment capital from the private sector for biotechnology has dried up,purely empirical attempts at stem cell therapy are difficult to justify,given the high probability of failure.In a1995report assessing the investment in gene therapy by the U.S.National Institutes of Health,a panel chaired by Stuart Orkin and Arno Motulsky recommended‘‘increased emphasis on research dealing with the mechanisms of disease pathogenesis,further development of animal models of disease,enhanced use of preclinical gene therapy approaches in these models,and greater study of stem cell biology in diverse organ systems’’(/oba/rac/panelrep.pdf).Similar recom-mendations regarding the need for proper investments in funda-mental aspects of stem cell therapeutics seems warranted and prudent at this time.Stem Cell Therapeutics:Frontline Clinical Trialsand Medical InnovationsA search of the Unites States government-sponsored website with the term‘‘stem cells’’lists over 4,000past,current,and anticipated trials,with over1,750now open(Figure1).The vast majority of open trials aim to build upon decades of research and clinical experience in hematopoi-etic transplantation(>1,200),and include strategies to expand the suboptimal dose of HSCs within umbilical cord blood,to complement gene defects in HSCs through viral transgene delivery(‘‘gene therapy’’),and to engineer T cells to attack malig-nancy via adoptive immunotherapy.Despite the relatively primi-tive understanding of therapeutic mechanisms for other stem cells,hundreds more trials are testing mesenchymal(115), adipose-derived(36),and neural stem cells(280),sometimes in quite bold and unconventional ways that bear little resem-blance to the known differentiation potential or modes oftissue Figure1.Clinical Trials of Major Stem Cell TypesPie chart indicating the relative numbers of open trials testing clinical inter-ventions for hematopoietic,neural,mesenchymal,adipose,and embryonic stem cells,as listed on the U.S.NIH website .Cell Stem Cell10,June14,2012ª2012Elsevier Inc.741regeneration or repair associated with these classes of stem cells.As of this writing,three trials pertain to products derived from ESCs.A wide array of stem cell studies are being carried out on a global basis on all continents,suggesting widespread clinical interest (Figure 2).Mesenchymal stem cells (MSCs)are defined by their fibro-blast-like morphology,adherence to plastic,expression of a specific set of surface antigens (CD105+,CD90+,CD73+),and capacity for osteogenic,chondrogenic,and adipogenic fates in vitro.MSCs are most often derived from bone marrow but can also be isolated from adipose tissue;adipose-derived stem cells may also consist of pericytes or endothelial progeni-tors that may differ somewhat in their properties from MSCs.Easy access to large quantities is an advantage for adipose-derived stem cells,which are being tested for soft-tissue repair and regeneration (Tobita et al.,2011).Both autologous (self)and allogeneic (foreign)MSCs are being tested in vivo to enhance healing that reflects their in vitro potential to form bone or cartilage,as in bone fracture and joint cartilage repair (Griffin et al.,2011).Although such studies are founded on strong preclinical evidence and sound scientific and clinical hypoth-eses,evidence for robust clinical efficacy of MSCs for ortho-pedic indications has been challenging to confirm,and to date no therapy based on MSCs has yet won approval by the U.S.Food and Drug Administration (FDA).The difficulty in proving the efficacy of regenerative treatments based on the well-char-acterized cellular potentials of MSCs suggests that our under-standing of how even familiar stem cells can be exploited therapeutically in vivo remains primitive.MSCs are being tested in a wide range of clinical indications where the clinical hypotheses are more speculative,the thera-peutic mechanisms are incompletely defined,and in some instances the preclinical evidence is highly contentious.For example,from a scientific foundation that can be traced to a highly controversial report that whole bone marrow would regenerate cardiac muscle following transplantation into injured hearts (Orlic et al.,2001),an observation later disproven (Balsam et al.,2004),thousands of patients have been treated in trials worldwide with various cell preparations of bone marrow or MSCs,with the scientific community debating the significance of the results (Choi et al.,2011).Subsequent studies have invoked a variety of contingent mechanisms including salutary paracrine effects on resident cardiomyocytes and putative cardiac stem cells,neoangiogenesis,and biomechanical alterations due to scarring (Gnecchi et al.,2008;Menasche,2011;Williams et al.,2011).The questions about underlying mechanism notwith-standing,combined meta-analyses of numerous trials has argued for measureable yet quite modest therapeutic effects,which has left practitioners unsure of the significance and robust-ness of these therapeutic approaches (Tongers et al.,2011).MSCs have also been widely tested for their capacity to mitigate autoimmunity,following somewhatserendipitousFigure 2.Worldwide Experimental Trials of Stem Cell-Based TherapiesWorld map showing locations of open,closed,and pending clinical trials of stem cell-based interventions as listed on .The relative numbers of trials performed outside of the U.S.may indeed be markedly understated because of reporting bias at the ernment clinical trials website.742Cell Stem Cell 10,June 14,2012ª2012Elsevier Inc.observations that MSCs can interfere with in vitro immunological assays such as mixed lymphocyte reactions and modulate production and function of the major classes of immune cells (Kode et al.,2009;Shi et al.,2011).Although it is unclear whether immune antagonism reflects any native function of MSCs in vivo, ex vivo expanded preparations have been infused in patients in hopes of mitigating transplant-related graft versus host disease and autoimmune conditions like Crohn’s disease,multiple sclerosis,and systemic lupus(Kebriaei and Robinson,2011; Shi et al.,2011).One canfind numerous reports of efficacy in the literature,but these are mixed with negative data(Kebriaei and Robinson,2011).The precise role of MSCs as agents for immune modulation remains to be proven.When clinical indications stray yet further from the presump-tive core functions of MSCs,and therapeutic mechanisms become increasingly speculative,clinical translation is a largely empirical rather than a rational effort.Likewise,while umbilical cord blood(UCB)has emerged as a viable alternative to other sources of HSCs(e.g.,mobilized peripheral blood or bone marrow)for the treatment of leukemia and nonmalignant hema-tologic conditions(Rocha et al.,2004),it has also become a common source for experimental interventions in a wide variety of nonhematologic indications as disparate as myocardial infarc-tion,multiple sclerosis,amyotrophic lateral sclerosis,cerebral palsy,traumatic brain injury,stroke,and inherited metabolic disorders(Copeland et al.,2009;Harris,2009;McKenna and Sheth,2011;Prasad and Kurtzberg,2009).Evidence exists that a number of distinct cell types can be cultured from UCB, including multipotential stem cells(Ko¨gler et al.,2004;Pelosi et al.,2012),but it is unclear whether such expandable cell pop-ulations exist at appreciable levels in unmanipulated samples. While in theory such cells could mediate therapeutic effects, nonhematologic indications for UCB transplantation have not been widely accepted into standard practice.When clinical investigation proceeds largely empirically,and without a deeper understanding of the basic therapeutic mechanisms,it is difficult to reformulate therapeutic strategies after clinical failures. Neural stem cells(NSC)can be cultured from fetal and adult brain and demonstrated to differentiate into neurons,oligoden-drocytes,and astrocytes in vitro.Given the wide array of neuro-logic conditions that have devastating clinical consequences, there is considerable interest in the therapeutic potential of neural regeneration therapies.However,neurodegenerative diseases,catastrophic stroke,traumatic brain injury,and spinal paralysis are among the most daunting challenges for regenera-tive medicine.The development of the brain and peripheral nerves and their interconnectedness with tissues throughout the body requires a remarkably complex choreography during fetal development.The proper milieu for directing the formation of highly specified neuronal subtypes and guiding their projec-tion to and interconnectedness with critical targets is highly unlikely to exist in the adult body.But faced with compelling unmet medical need and desperation on the part of patients, there are hundreds of investigator-initiated clinical trials occur-ring in academic settings(Figure1),and several companies have forged efforts to develop novel therapies through intracere-bral or spinal transplantation of neural stem cells(Trounson et al., 2011).StemCells Inc(California,USA)has tested NSCs in Batten’s disease(neuronal ceroid lipofuscinosis)and was able to document safe delivery but discontinued the trial because of the inability to accrue an adequate number of patients.Their current focus is Pelizaeus-Merzbacher disease,a myelin disorder,and chronic spinal cord injury.Other companies are testing NSC transplant for stroke(ReNeuron,United Kingdom), amyotropic lateral sclerosis(Neuralstem,Inc,Maryland,USA), and Parkinson’s disease(NeuroGeneration,California,USA).In most of these cases,the clinical hypotheses being tested do not depend upon the generation of neurons de novo,but instead on complementation of enzyme deficiencies,remyelination,or modulation of endogenous repair through neoangiogenesis or neuroprotection.Although widely publicized,there are comparatively few clin-ical trials of products derived from human embryonic stem cells (hESCs).Thefirst trial conducted in humans delivered oligoden-drocyte progenitors for the remyelination of spinal cord axons damaged through crush injury.These studies were based on extensive preclinical experience with the derivation and charac-terization of oligodendrocytes and their delivery in animal models that showed remyelination and restoration of motor function (Keirstead et al.,2005;Liu et al.,2000;McDonald and Belegu, 2006;McDonald and Howard,2002;McDonald et al.,1999; Nistor et al.,2005).Moreover,thisfirst trial required a herculean effort to satisfy FDA regulatory oversight,by report entailing the submission of over20,000pages of data and documentation. The trial,sponsored by the Geron Corporation(California, USA),enrolled and treated itsfirst four patients before being dis-continued due to a decision by company management to focus on alternative corporate priorities(Baker,2011).No formal results have yet been released regarding the phase1clinical trial in thisfirst small cohort of patients,but the primary endpoints were safety of the cells,and at the very least one hopes that some evidence will be gleaned that products of ESCs can be delivered without risk of teratoma,although long-term follow-up of all treated patients will be necessary.The only other current clinical trials involve transplantation of hESC-derived cells to treat retinal blindness.This condition takes many forms,both genetic and age-related,and as a group of disorders has many appealing features for stem cell-based interventions.The retina is accessible for local delivery of cells, which can then be monitored via direct visualization.The retina may also provide some degree of immune privilege.Very prelim-inary results of a trial involving the subretinal injection of hESC-derived retinal pigment epithelial cells for Stargardt’s macular degeneration and another for age-related macular degeneration sponsored by the company Advanced Cell Technologies(ACT) were recently reported,despite experience on only one patient in each trail(Schwartz et al.,2012).Only one of the two patients showed evidence of persistent cells but both were reported to show some restoration of visual perception.While it is difficult to draw conclusions from these early trials due to the limited numbers of patients involved and the very brief4month period of follow-up,the trials represent milestones in that the investiga-tors succeeded in clearing considerable regulatory hurdles and met very high standards of preclinical cell characterization and quality control prior to exposing patients to the risk of ESC-based products.The experience alone,for both investigators and regulators,is an essential albeit small step forward in the long path to establishing ESC-based therapeutics.Cell Stem Cell10,June14,2012ª2012Elsevier Inc.743While MSCs,NSCs,and products from ESCs are being tested in the context of numerous clinical trials,yet another arm of regenerative medicine—tissue engineering—is comingling MSCs or a variety of other cultured cell types with biocompatible materials to solve surgical challenges.Reconstruction of blad-ders(Aboushwareb and Atala,2008;Atala,2011;Tian et al., 2010),tendons(Sun et al.,2011),and complex structures like the trachea(Macchiarini et al.,2008)represent solutions to highly personal needs of specific patients and are acceptably performed as highly innovative and individualized surgical thera-pies,part of the long tradition of surgical innovation.The mech-anisms for developing such novel interventions and gaining acceptance by the surgical and biomedical communities involve the same core principles required for medical interventions—sound scientific rationale and methods,institutional and practi-tioner accountability,thorough and rigorous informed consent, patient follow-up,timely reporting of adverse events,peer review of therapeutic claims,and publication in the medical literature. The potential for therapeutic innovation at the interface of stem cell biology and tissue engineering is particularly appealing but beyond the scope of this review.I refer the reader instead to excellent recent reviews(Griffin et al.,2011;Peck et al.,2012; Sun et al.,2011).Anticipated Future Interventions and Opportunities Among the many disparate conditions,disorders,and diseases for which stem cells have offered promise,a few stand out as particularly compelling.In general,they are conditions where defects are largely cell autonomous and entail the loss or dysfunction of a single class of cells or a monocellular compo-nent of a complex tissue,such that restoration of function through cell replacement would be curative or significantly ameliorate symptoms.Those conditions most amenable to treat-ment present the least anatomic complexity and affect tissues that do not typically regenerate spontaneously because they lack endogenous pools of tissue stem cells.We can predict ulti-mate success with most confidence if some clinical evidence already exists that cell replacement might indeed be therapeutic, for instance through prior assessments of cadaveric or fetal tissue transplantation.For conditions previously treated with cadaveric or fetal material,efficacy may be limited by the inade-quate supply or quality of the cells,making pluripotent or reprog-rammed cell sources advantageous.Parkinson’s Disease.Although neurologists recognize that Parkinson’s disease(PD)has systemic features,the chief deficit remains the loss of a specific subtype of midbrain dopaminergic neurons located in a deep brain structure,the substantia nigra, whose many connections to the striatum are responsible for regulating movements,such that PD patients suffer from immo-bility,rigidity,and tremor.Drug replacement with precursors of dopamine(DA),dopamine agonists,or antagonists of dopamine metabolism serves to ameliorate symptoms but cannot stem the inexorable decline in most patients.Based on decades of expe-rience from several groups with transplantation of fetal tissue sources of DA neurons,deep brain transplantation can indeed restore local DA production and ameliorate symptoms,with some patients showing durable improvement and graft integrity after two decades(Freed et al.,1992;Lindvall et al.,1990;Lind-vall et al.,1994;Piccini et al.,1999,2005).Functional imaging and postmortem analysis support the stable integration and persistence of grafts in some patients,prompting continued enthusiasm for this approach among some practitioners, provided that a suitable source of DA neurons can be defined (Freed et al.,1992;Lindvall et al.,1990,1994;Ma et al.,2010; Nakamura et al.,2001;Piccini et al.,1999,2000).Others, however,remain skeptical,in part because a trial of fetal grafts randomized against sham surgery was inconclusive,with some patients sustaining functional decline postsurgery due to dyski-nesias as a result of excessive graft function(Freed et al., 2001).Supporters of cell therapy for PD point out that a more reli-able,consistent,and defined source of DA neurons would justify further testing of transplantation strategies.Many groups have differentiated DA neurons from both neural stem cell and pluripotent stem cell sources and proven func-tional in rodent models(Hargus et al.,2010;Sanchez-Pernaute et al.,2008;Tabar et al.,2008;Wernig et al.,2008).Analysis of this DA neuron production has not always distinguished among the many different classes of neurons that produce DA throughout the neuraxis,but recent advances have made possible the differentiation from pluripotent cell sources of regionally specific midbrain DA neuronal subtypes whose defi-ciency is most affected in PD is possible,and such cells have been documented to function in rodent and primate models (Chambers et al.,2009;Fasano et al.,2010;Kriks et al.,2011). Moreover,techniques for producing personalized autologous stem cells via somatic cell reprogramming now exist,and it has been shown that autologous cells function better than cells derived from unrelated donors in rodent models of PD transplant (Tabar et al.,2008).The availability of highly specified,defined, autologous DA neuron preparations creates legitimate opportu-nities for testing in PD patients,including the testing of specific doses to establish a dose-response curve.Nevertheless,even optimistic accounts identify the significant hurdles that remain (Lindvall and Kokaia,2010).Notably,any cell therapy must ulti-mately be superior in safety and efficacy to any drug therapy, and establishing such utility will require large-scale and pains-taking prospective trials to be conducted over many years. Thus,despite promise,cell therapy as the standard of care for PD is but a distant horizon.Cell therapy for PD will need to be efficacious and safe to compete with the highly effective drug treatments that currently exist(Hjelmgren et al.,2006).In contrast,a condition like Huntington’s disease,which has no viable drug therapy and is invariably fatal,is an appealing alternative therapeutic target for cell transplantation therapies derived from NSCs and ESCs. Intrastriatal transplantation of homotypic fetal tissues has shown graft durability and reports of amelioration of symptoms in HD patients(Gallina et al.,2010;Nicoleau et al.,2011).As for PD, an improved cell source would facilitate the necessary studies to optimize the dose and target region for cell transplantation. Techniques for directed differentiation of ESCs into relevant medium spiny neurons and amelioration of rodent models of HD have been reported and bode well for future translational clinical studies(Benraiss and Goldman,2011). Autoimmune Diabetes Mellitus.Type1diabetes(T1D;insulin-dependent,juvenile onset)is an autoimmune condition that involves active immune destruction of the beta cells of the islets of Langerhans of the pancreas,leaving the patient with inade-quate supplies of insulin and susceptibility to hyperglycemic744Cell Stem Cell10,June14,2012ª2012Elsevier Inc.。
Nature子刊：T细胞通过外泌体转移DNA起始树突状细胞的免疫反应大昌华嘉Particle Metrix，纳米颗粒跟踪分析仪西班牙研究人员最新在 Nature Communications 上发表文章，报道了对病原体（如病毒和细菌）反应早期阶段有关免疫系统防御的机制。
该研究结果有助于理解早期阶段开始的细胞过程，并解释了免疫系统的不同细胞群如何交流以对病原体产生有效反应。
CNIC研究人员证明，纳米囊泡中含有的线粒体DNA会激活受体细胞的警觉状态，从而激活抗病毒遗传程序。
这些称为外泌体的纳米囊泡由T淋巴细胞产生，并通过细胞间接触被树突细胞吸收。
针对病原体的免疫应答需要T淋巴细胞和抗原呈递细胞，特别是树突细胞之间通过免疫突触的形成进行特异性物理相互作用。
在此过程中，细胞通过细胞表面的受体-配体接触和外泌体的转移交换信息。
迄今为止的研究主要集中在免疫突触如何激活T细胞中的信号传导途径；相反，树突细胞接收的信号的特性和效果受到的关注相对较少。
该研究由Francisco Sánchez-Madrid教授领导的小组进行，该小组是CNIC细胞间通讯实验室的首席研究员，Prince Princesa医院的免疫学服务负责人，以及马德里自治大学的免疫学教授。
在以前的工作中，该小组证明T细胞可以在免疫突触形成过程中将外泌体转移到树突细胞。
Sánchez Madrid教授解释说，在这项新研究中，研究小组描述了这些纳米囊泡如何“转运线粒体来源的DNA和蛋白质”。
该研究揭示线粒体组分被导向T细胞中的内体系统，在胞内体中外泌体形成并随后分泌，证明了内体和线粒体区室之间的紧密关系。
纳米膜泡中存在的DNA“直接负责通过cGAS/STING途径增加抗病毒基因的表达，从而检测到细胞核外的DNA。
”正如研究作者Daniel T orralba所解释的那样，“进口的DNA可以作为一种警报，触发免疫系统激活抗病毒反应。
下一代免疫疗法即将问世，体内细胞重编程技术有望引领潮流2022-08-05 10:07·医学营养治疗原创药明康德药明康德 2022-08-05 07:30 发表于美国编者按：在传统的小分子药物和抗体药物之外，细胞和基因疗法、RNAi和其它寡核苷酸疗法、CRISPR基因编辑疗法等更多新型的分子类型在近年逐渐走向前台，成为了生物医药产业的关注焦点，有望改写患者们的未来治疗格局。
生物医药迈入崭新时代之际，药明康德内容部也已启动“迅猛新分子”系列，邀请新分子疗法汹涌浪潮的弄潮儿进行访谈——这些访谈大咖执掌的公司都专注于开创全新类型的疗法，并在近期完成了大额早期融资，可谓是产业中冉冉升起的未来之星。
在药明康德内容部的系列访谈里，他们也将向产业介绍如何使用新型分子类型突破现有疗法的局限，带来崭新的突破！嘉宾简介：本期访谈的嘉宾Daniel Getts博士是Myeloid Therapeutics公司的联合创始人和首席执行官。
Myeloid Therapeutics公司是一家临床阶段的mRNA免疫疗法公司，主要利用髓系细胞来设计新的疗法，通过引发机体中广泛的免疫反应来治疗癌症和自身免疫性疾病。
Daniel Getts博士有着多年医药行业经验，曾先后担任Tolera Therapeutics、Cour Pharmaceuticals、TCR2 Therapeutics等多家医药公司的研发负责人，在新药靶点开发、开展临床前研究与转化医学项目等方面积累了丰富的经验，并在同行评议的学术期刊中发表了超过45篇研究，其中多篇论文发表在Nature Biotechnology, ScienceTranslational Medicine等知名学术期刊。
在药明康德内容部的系列访谈里，他将与我们分享他对于细胞免疫疗法研发的洞见。
药明康德内容部：Daniel，感谢您抽空接受我们的访谈！针对癌症和自身免疫性疾病疗法的研发，在您看来医药行业所面临的挑战有哪些，以及您的公司相应的对策是什么？Daniel Getts博士：Myeloid公司主要针对疑难、并且具有高度未竟医疗需求的病症，比如外周T细胞淋巴瘤（PTCL）、肝癌和胶质母细胞瘤。
肝动脉注射锰锌铁氧体磁性纳米颗粒治疗兔VX2肝癌王子妤;王丽;余辉;卢勤;张东生【期刊名称】《中国组织工程研究》【年(卷),期】2011(015)034【摘要】BACKGROUND: Compared with traditional thermotherapy method, magnetic fluid hyperthermia (MFH) has good magneticresponse, and which can achieve automatic temperature control and targeting of tumor thermotherapy in certain high frequencyalternating magnetic field.OBJECTIVE: To prepare Mn-Zn ferrite (MZF) magnetic nanoparticles, and to observe the inhibition effects of MZF magneticnanoparticles combined with hepatic artery interventional therapy on rabbit VX2 liver tumor.METHODS: Rabbit VX2 liver tumor models were established by implanting tumor piece and cell suspension via abdominal incision.At 14 days after modeling, a total 24 rabbits were randomly divided into 4 groups as follows: control group (sodium chloride), MZFgroup, MZF hyperthermia group, adriamycin gro up, each groups contain 6 VX2 rabbits. The rabbit hepatic proper artery wasselectively catheterized by 3F micro -catheters via right femoral artery. The rabbits of MZF hyperthermia group were exposed toalternating magnetic field for three times. At 14 days after interventional operation, the tumor size of liver tissue was detected andharvested for histopathology examination.RESULTS AND CONCLUSION: MZF nanoparticles were approximately global and their average diameterwas 20-30 nmexamined by transmission electron microscope (TEM), and they had good magnetic responsiveness under alternating magneticfield. At 14 days after treatment, large areal necrosis arose in tumor in MZF hyperthermia group, the tumor-inhibiting rate was70.84％ higher than the other groups, and the difference was significant (P ＜ 0.05 or P ＜ 0.01). MZF nanoparticles can absorbelectromagnetic waves into heat, through interventional therapy which can significantly inhibit the growth of rabbit VX2 liver tumor.%背景:与传统的热疗方法相比,磁流体热疗具有很好的磁响应性,在一定高频交变磁场下能实现肿瘤热疗的自动控温和靶向治疗等优点.目的:制备锰锌铁氧体磁性纳米颗粒,观察其介入治疗兔VX2 肝癌的效果.方法:采用开腹后瘤粒悬浮液针头注入法制作兔VX2 肝癌模型,造模后14 d随机数字表法分为对照组(生理盐水)、锰锌铁氧体磁性纳米颗粒非热疗组、锰锌铁氧体磁性纳米颗粒热疗组、阿霉素组,均采用3F导管从右侧股动脉选择至肝固有动脉动脉注入药物后拔管.锰锌铁氧体磁性纳米颗粒热疗组于介入后行热疗3次.介入治疗后14 d取肝脏组织测量肿瘤大小,并做病理组织学检查.结果与结论:透射电镜下观察制备的锰锌铁氧体磁性纳米颗粒为球形,大小为20~30 nm,在交变磁场下有良好的磁感应升温能力.治疗后14 d,锰锌铁氧体磁性纳米颗粒热疗组肿瘤大面积坏死,肿瘤抑制率达到70.84%,明显高于锰锌铁氧体磁性纳米颗粒非热疗组、阿霉素组与对照组(P < 0.05或P < 0.01).说明锰锌铁氧体磁性纳米颗粒可吸收电磁波转化为热能,通过介入治疗可显著抑制兔VX2 肝癌生长.【总页数】4页(P6360-6363)【作者】王子妤;王丽;余辉;卢勤;张东生【作者单位】东南大学医学院,江苏省南京市,210009;南京中医药大学基础医学院,江苏省南京市,210046;东南大学医学院,江苏省南京市,210009;东南大学医学院,江苏省南京市,210009;东南大学医学院,江苏省南京市,210009;东南大学医学院,江苏省南京市,210009【正文语种】中文【中图分类】R318【相关文献】1.肝动脉门静脉化疗栓塞治疗兔VX2肝癌后残癌新生血管的研究 [J], 吴鸿峰;余梁;胡茂能2.SPIO纳米药物载体联合碘化油经肝动脉栓塞治疗兔VX2肝癌 [J], 梁琪;邓灵灵;冯智超;刘晓;丁劲松;胡鹏志;王维3.经肝动脉碘化油/无水乙醇混合剂栓塞消融术治疗兔VX2肝癌的实验研究 [J], 钱亭;陈茂振;高峰;尹化斌4.肝动脉臭氧化碘油栓塞治疗兔VX2肝癌的初步研究 [J], 张学彬;花迎雪;仇晓霞;张冰星;路青;沈加林;许建荣5.经肝动脉灌注DC-B载药微球对照碘化油治疗兔VX2肝癌模型对VEGF的影响[J], 刘敦;陈臣;帕提玛·阿布力米提因版权原因，仅展示原文概要，查看原文内容请购买。
Ana I Flores, Gonzalo J Gómez-Gómez, Ángeles Masedo-González, M Pilar Martínez-Montiel, Servicio de Aparato Digestivo, Hospital 12 de Octubre, Avda. de Córdoba, 28041 Madrid, Spain Author contributions: All authors contributed to this work. Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: / licenses/by-nc/4.0/Correspondence to: M Pilar Martínez-Montiel, MD, Servicio de Aparato Digestivo, Hospital 12 de Octubre, Avda. de Córdoba, s/n, 28041 Madrid, Spain. pilarmarmon123@ Telephone: +34-91-7792667Received: July 29, 2014Peer-review started: July 29, 2014First decision: August 28, 2014Revised: October 30, 2014Accepted: November 7, 2014Article in press: November 10, 2014Published online: March 26, 2015AbstractInflammatory bowel diseases are inflammatory, chronic and progressive diseases of the intestinal tract for which no curative treatment is available. Research in other fields with stem cells of different sources and with immunoregulatory cells (regulatory T-lymphocytes and dendritic T-cells) opens up new expectations for their use in these diseases. The goal for stem cell-based therapy is to provide a permanent cure. To achieve this, it will be necessary to obtain a cellular product, original or genetically modified, that has a high migration capacity and homes into the intestine, has high survival after transplantation, regulates the immune reaction while not being visible to the patient’s immune system, and repairs the injured tissue.Key words:Mesenchymal stem cell; Hematopoietic stem cell; Inflammatory bowel disease; Crohn’s disease; Ulcerative colitis; Amniotic fluid stem cells; Induced pluripotent stem cells; Intestinal stem cells; Endothelial progenitor cells; Tolerogenic immune cell therapies© The Author(s) 2015. Published by Baishideng Publishing Group Inc. All rights reserved.Core tip: Inflammatory bowel diseases are inflammatory, chronic and progressive diseases of the intestinal tract.A limited experience is available with hematopoietic and mesenchymal stem cell transplantation for the treatment of these conditions. Research is ongoing with other cell lines which have been used in conditions alike to inflammatory bowel disease and which will possibly have a therapeutic role in this condition.Flores AI, Gómez-Gómez GJ, Masedo-González A, Martínez-Montiel MP. Stem cell therapy in inflammatory bowel disease: A promising therapeutic strategy? World J Stem Cells2015; 7(2): 343-351 Available from: URL: http://www.wjgnet. com/1948-0210/full/v7/i2/343.htm DOI: http://dx.doi. org/10.4252/wjsc.v7.i2.343INTRODUCTIONInflammatory bowel disease (IBD) mainly consists of two clinical conditions, Crohn’s disease (CD) and ulcerative colitis (UC). It is mainly characterized by chronic, destructive inflammation of the gastrointestinal tract for which no curative treatment is currently available.Its etiology is unknown, but it is accepted that it could be the result of loss of tolerance to intraluminal bowel antigens[1]. Genetic, environmental, and microbiological factors are involved in its development,REVIEWSubmit a Manuscript: /esps/ Help Desk: /esps/helpdesk.aspx DOI: 10.4252/wjsc.v7.i2.343World J Stem Cells 2015 March 26; 7(2): 343-351ISSN 1948-0210 (online)© 2015 Baishideng Publishing Group Inc. All rights reserved. Stem cell therapy in inflammatory bowel disease: A promising therapeutic strategy?Ana I Flores, Gonzalo J Gómez-Gómez, Ángeles Masedo-González, M Pilar Martínez-Montieltogether with morphological and functional changes in the intestinal barrier associated to an impaired immune response[2]. Early data supporting genetic involvement in the pathogenesis of IBD come from familial clinical studies showing a greater incidence in twins[3,4], first-degree relatives[5,6] and given ethnic groups[7,8]. Genome-wide association scan studies have allowed for identification of more than 163 loci associated to IBD[9], 73 genes associated to CD and 47 to UC[10], and overlapping genes for both conditions have also been found[11]. Genetic factors would however account for less than 25% of cases[12]. The exception is represented by a monogenic disorder referred to as IBD-like diseases, which are associated with severe colitis in childhood and have at most three loci alternatives[13]. On the other hand the increase of the incidence of IBD suggests that environmental factors are more important than genetic factors in the development of IBD[14].Since IBD etiology is currently unknown, current treatment is intended to control the inflammatory intestinal process, thus avoiding irreversible structural damage. However, current therapeutic results are discouraging. Thirty-three percent of patients with CD do not respond to anti-TNF alfa therapy[15-18], and one third of responders loss the response[19]. Based on all the foregoing and on advances in understanding of the pathophysiological mechanisms involved in IBD development, new biological drugs and cell therapies are being investigated.Future of the cellular-based therapy in IBD: Lessons from preclinical and clinical studiesCell therapies are promising candidates for the treatment of IBD. However, inconsistent results have emerged from current clinical trials using both, hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). To establish the best stem cell type, the administration route and optimal dose of cells to achieve an effective therapy and to guarantee the safety of the patient, in-depth basic research is necessary. Therefore, preclinical studies using different animal models are necessary to understand the pathogenesis of IBD. These studies will facilitate a better design of preclinical stem cell therapies that will eventually become a suitable clinical therapy for IBD patients.ADULT STEM CELLSAt present, HSCs and MSCs have been used in several clinical trials. However, up to now, the results are unpredictable. For additional information, the reader is referred to the recent review published by our group in 2014[20].Hemapoietic stem cells transplantationUse of hematopoietic stem cells transplantation (HSCT) in IBD is restricted to severe CD with few therapeutic options. These are patients who do not respond to standard treatment in whom surgery is not an option due to extent of disease. HSCT may also be used in monogenic diseases, such as interleukin-10 deficiency, where allogenic bone marrow transplant would correct the disease by reconstituting a new immune system.Allogenic HSCT is not currently accepted for the treatment of CD because of its high mortality rate. Current studies focus on autologous HSCT, which is intended to “reset” the immune system of the patient. Once reactive T-lymphocytes and memory cells of the patient are eliminated by chemotherapy (lymphoablation), administration of autologous HSCs generates an immunotolerant system[21-23]. Unfortunately, this effect will probably be transient until the patient cells start the inflammatory mechanisms again.Clinical experience is limited (T able 1), with the Burt study[25]reporting complete remission in all their 24 patients. We are currently waiting for the results of the ASTIC study[28]. This is a phase Ⅲclinical trial comparing two treatment arms intended to clarify whether improvement is due to reset of the immune system or to transplantation. The first arm uses chemotherapy followed by early transplantation (30 d), and the second arm chemotherapy with late transplantation (13 mo). Results reported to date include a high adverse effect rate and appear to suggest efficacy of transplantation. Mesenchymal stem cell transplantationMSCs seem to be a promising therapeutic strategy for IBD because of their ability to selectively home in to injury/inflammation areas after systemic administration, and their immunosuppressive and tissue healing properties[30,31]. However, the clinical data published about MSCs transplantation in IBD patients showed conflicting results. An explanation for these inconsistent results could be the different sources used to obtain the MSCs that could have distinct differentiation and regeneration capabilities and the variety of protocols used for their isolation and culture. A better understanding of the MSCs biology and mechanisms of action and the exploration of other sources of stem cells in preclinical models of IBD are necessary.Bone marrow and adipose tissue are the main sources of MSCs for both experimental and clinical studies. However, these sources have several disadvantages such as the invasive procedure used for their isolation, the small number of MSCs isolated, and the low proliferation and differentiation capacities related to donor age. For this reason, in the last few years the search for alternative tissue sources for MSCs has become of vital importance. Placental tissues, both fetal (amniotic fluid, Wharton’s jelly, amniotic membrane, chorionic villi) and maternal (decidua) represent an important source of MSCs with some advantages including the isolation of large number of cells in a non invasive way[32,33]. In addition, likeFlores AI et al. Stem cell in inflammatory bowel diseasebone marrow MSCs, placenta-derived MSCs are non-immunogenic and immunomodulatory stem cells with high expansion and differentiation capacity[32-35].An important issue in using MSCs is their safety. Although some studies supported that there is a risk of MSCs malignant transformation[36,37], several recent studies using different types of MSCs supported that there is neither in vitro risk of development chromosomal aberrations after long term culture nor in vivo induction of tumors[30,32,38,39].For IBD treatment it is essential to increase the number of cells that migrate and home in to the intestine. A preclinical model of radiation enteritis treated with MSCs genetically modified to express the CXCR-4 receptor showed an increase of MSCs migration to intestinal site of injury and an improvement of symptoms[40]. In the same way, MSCs coated with antibodies against vascular cell adhesion molecule VCAM-1 showed an increased cell migration of MSCs to inflamed colon and thereby an increased tissue repair capacity[41]. A different strategy is to select a subpopulation of MSCs within the bone marrow that expresses high levels of EphrinB2. This subpopulation has an increased migration capacity to intestinal injury areas, and as a consequence, these MSCs would help to improve healing of intestinal injury[42]. Once MSCs engraft in the intestinal damaged tissue they can proliferate and transdifferentiate into intestinal stem cells, or secrete cytokines and growth factors that will promote the proliferation and differentiation of intestinal stem cells in order to repair the injured areas of the intestinal tissue[43].Besides the migration, homing and tissue repair capabilities of MSCs, they also have an important function in modulating the inflammation and high immune response within the injured tissues. These immunomodulatory properties of MSCs are of special importance in the treatment of IBD. Systemic administration of bone marrow MSCs in a mouse model of chemical-induced colitis[43] and in a pig model of radiation-induced proctitis[44], down-regulated autoimmune and inflammatory responses, and as a consequence, facilitated tissue regeneration.The experience in luminal CD is limited (T able 2). Experience in UC is even smaller, and was mainly obtained in Russian studies about response of clinical activity[50], changes in the pattern of systemic cytokines[51] and elimination of cytomegalovirus after Mesenchymal stem cell transplantation (MSCT)[52]. The most important work in this field is a phase Ⅲ study[48] that plans to include 330 patients who will be treated with MSCs at different doses, but final results are not expected until 2018. According to data reported to date, the safety profile appears to be favorable, and formation of aberrant tissue has not been detected.As regards local treatment for perianal CD (T able 3), a single study using bone marrow cells is available[55], and there is an 11-year experience of the Spanish group with MSCs taken from fat tissue (ASCs)[53,54,56], initially autologous, except for a phase Ⅰ/Ⅱ trial using donor cells[56]. We are currently waiting for completion of a phase Ⅲ trial using donor cells which is planned to recruit a large patient sample. T wo Korean studies using autologous ASCs have more recently been published. The first was carried out to evaluate the safety of the treatment[57]. The second is a phase Ⅱstudy[58].A tot al of 43 patients were injected with ASCs. Among these, 33 were included in the modified per protocol analysis. The results showed complete sealing of 27 patients 8 wk after the final injection ofNA: Not available; CDAI: Crohn´s disease activity index; IQR: Interquartile range; CMV: Cytomegalovirus.Flores AI et al. Stem cell in inflammatory bowel diseaseRecently , AFSCs were used in a neonatal rat model of necrotizing enterocolitis, one of the primary causes of morbidity and mortality in neonates, showed a decrease in intestinal damage, an increase in gut tissue repairand a higher survival [59,60]. A better understanding of the AFSCs biology and mechanisms of action may help to develop strategies for their use in other IBD.INDUCED PLURIPOTENT STEM CELLSThese are pluripotent cells derived from somatic cells by the introduction of reprogramming factors (Oct-4, Sox2, Kfl4, c-Myc, Nanog and Lin28). These pluripotent cells can be differentiated to anyASCs. No serious adverse effects were reported.It is obvious that MSCs are a promising tool in the treatment of IBD. However , a large amount of work remains to be done to understand the mechanisms through which MSCs regulate the immune system, homeostasis and tissue repair . This knowledge will provide us with new tools to implement an effective MSCs-based treatment for IBD.AMNIOTIC FLUID STEM CELLSAmniotic fluid stem cells (AFSCs) are isolated from the excess of second-trimester amniotic fluid obtained during routine amniocentesis for prenatal diagnosis.BM: Bone marrow; SAEs: Serious adverse events; NA: Not available; CD: Crohn´s disease; UC: Ulcerative colitis.Table 3 Mesenchymal stem cell transplantation studies in perianal Crohn's diseaseBM: Bone marrow; CD: Crohn´s disease; NA: Not available.Flores AI et al . Stem cell in inflammatory bowel diseasetissue specific cells to generate autologous cells for cell-replacement therapy[61]. Human intestinal organoids have recently been generated from these cell lines[62]. This will allow in the future for studying the pathophysiology of the disease and for testing new therapies, including generation of potentially viable tissues. Induced pluripotent stem cells (iPSCs) have been derived from somatic cells obtained from patients suffering a variety of diseases and important progress has been made in establishing preclinical iPSC-based disease models including IBD[63]. Although iPSCs do not have the ethical problems of embryonic stem cells, there are many similarities between them and, as a consequence, iPSCs could develop teratomas following transplantation, hindering their use in clinical trials. INTESTINAL STEM CELLSIntestinal stem cells (ISCs) are a rare population of fast-cycling Lgr5+cells and slow-cycling Tert+/ Bmi1+ cells situated above them at the crypt base. ISCs are in charge of the renewal of the intestinal epithelium which is changed every 4-5 d and in the regeneration of the intestinal epithelium after injury or inflammation[64]. It has been suggested that Lgr5+ cells and Tert+/Bmi1+ cells are two functionally different populations of ISCs[65]. Lgr5+ cells are responsible for the maintenance of the normal homeostatis conditions, whereas Tert+/Bmi1+ cells are more quiescent cells responsible for the intestinal epithelium regeneration under injury or inflammation conditions[66,67]. ISCs have proliferation and mutipotency capabilities, i.e., they are able to divide and later differentiate into all intestinal subtypes (enterocytes, globet cells, Paneth cells and neuroendocrine cells). Recently, research in the ISCs field has advanced greatly and many ISCs markers have been identified[68]. However, an exhaustive characterization of ISCs as well as the identification of specific markers still remains elusive[64].T ransplantation of fetal and adult ISCs expanded in vitro presented a strong engraftment and healing potential in a colonic injury model in mice[69,70]. However, ISCs in culture maintained as single cells have a very limited use in the study of the development of IBD and as a method for drug screening. Recently, intestinal organoids have been obtained from adult mouse and human ISCs[66,71,72]. These organoids were able to engraft and repair murine and human epithelium and represent an important step forward in the treatment of IBD[73]. Human organoids will be a very useful tool to study the pathological mechanisms of the disease from a specific patient and to test which is the best treatment to repair the intestinal epithelium for that patient. These organoids will be an important way to reach a more personalized medicine for IBD. These results highlight that those intestinal stem cells are a very promising source of stem cells for future patient-specific regeneration of the digestive tract[68]. ENDOTHELIAL PROGENITOR CELLS Besides local inflammation, IBD is characterized by anomalous angiogenesis/vasculogenesis and severe damage in epithelial cells[74]. Important results have been obtained using endothelial progenitor cells (EPCs) for the treatment of hindlimb ischemia and myocardial ischemia. Recently, EPCs transplantation into fetal sheep showed an efficient migration and homing within the mucosal layer and a contribution to the vasculogenesis of the intestine[75]. These results suggest that EPCs could represent an additional source of cells for IBD cellular therapy, on their own or in combination with other stem cells such as MSCs. TOLEROGENIC IMMUNE CELL THERAPIES In inflammatory disorders, special interest has been given to therapeutic strategies that could enhance the patient´s tolerance response to intraluminal antigens. T-regulatory cells (T regs) suppress immune responses of other cells and maintain tolerance to self-antigens. T regs can be generated ex vivo by activation of both, murine and human CD4 T cells, suggesting that they could be an extra source of cells for cellular therapies in IBD. Intraperitoneal injection of induced T regs in a mouse model of chronic colitis showed an attenuation of the preexisting gut inflammation response[76,77].Dendritic cells (DCs) are antigen-presenting cells involved in immunity and tolerance. DCs seem to be the most important regulators of immune tolerance in the gastrointestinal system, however, extensive studies are necessary to understand their role in this tissue and their mechanisms of action[78]. Like MSCs, tolerogenic-DCs (tol-DCs) do not express neither MHCII nor the T-cell co-stimulatory molecules, and will not activate an immune response in the host. Ex vivo generated tolerogenic-DCS are available as a clinical grade product and used as therapeutic vaccines to restore antigen-specific tolerance in autoimmune diseases[78]. T ol-DCs have been used in very few recent clinical studies such as rheumatoid arthritis and other not inflammatory diseases, and as a result conclusions about their clinical efficiency are still elusive. Several mouse models of colitis showed an important effect of tol-DCs in the prevention and reduction of symptoms of IBD[79-83]. However, several questions must be resolved before tol-DCs can be used in IBD cellular therapy in humans, mostly due to the differences in IBD and tol-DCs between mice and humans[84]. CONCLUSIONThe goal for stem cell-based therapy is to provide a permanent cure for IBD. To achieve this, it will be necessary to obtain a cellular product (original or genetically modified) that has a high migration and homes into the intestine, has high survival after Flores AI et al. Stem cell in inflammatory bowel diseasetransplantation, regulates the immune reaction which is not detectable to the patient’s immune system, and will repair the injured tissue. Intestinal tissue is composed of several cell types and IBD are characterized by widespread damage. Cell-based therapies will probably be designed as a combination of several cell types that will produce a synergic therapeutic response. ACKNOWLEDGMENTSThe authors thank Mr. Ian Ure for the English grammar revision of this manuscript.REFERENCES1 Martínez-Montiel MP, Muñoz-Yagüe MT. Biologic therapies forchronic inflammatory bowel disease. Rev Esp Enferm Dig 2006; 98: 265-291 [PMID: 16792456]2 Neurath MF, Travis SP. Mucosal healing in inflammatory boweldiseases: a systematic review. Gut 2012; 61: 1619-1635 [PMID: 22842618]3 Tysk C, Lindberg E, Järnerot G, Flodérus-Myrhed B. Ulcerativecolitis and Crohn’s disease in an unselected population of monozygotic and dizygotic twins. A study of heritability and the influence of smoking. 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