chp 7 tour of cell

Gibco boxy bottle 2008–presentGibco One Shot 50 mL bottle2016–presentGibco square plastics1987–1996Gibco round plastics 1996–2007Gibco glass bottle 1962–1986For performance and consistency essential to successful cell cultureG ibco sera—committed to quality a nd innovation since 1962Cell cultureDelivering reliable cell culture productsfor over 60 yearsA history of innovationIn 1962, Leonard Hayflick made the important discovery that there is a finite capacity for normal human cells to replicate in culture. This finding overturned a long-held belief about the potential immortality of cultured cells and has had far-reaching implications in life science research. That same year, Bob and Earline Ferguson, two biologists working from their garage in Grand Island, New York, recognized the business potential of supplying animal sera for research use. From this humble beginning, Gibco™ sera rose to the forefront of products supporting global life science research. Gibco™ cell culture products are now an important part of Thermo Fisher Scientific.How did we become a world leader for Array sera, media, and reagents? The keyto the success of Gibco products hasalways been the consistent delivery ofquality, which helps reduce the number ofunknowns that scientists may experiencein their work. Across the global life sciencecommunity, Gibco products have areputation for reliability—allowing scientiststo focus on more important things thantroubleshooting cell culture problems. Inaddition to supporting innovators in lifescience research, Thermo Fisher Scientificis a leading supplier to the globalbiopharmaceutical industry. Part of oursuccess is due to our strong commitmentto both small and large laboratories,ranging from the research bench toproduction-scale facilities.The original manufacturing site located inGrand Island, New York, is now just oneof many manufacturing facilities worldwidethat produce Gibco cell culture products.Through our commitment to quality, wecontinue to provide scientists with theconsistent reliability, service, value, andinnovation that have made Gibco productsa global market leader for over 50 years.The right sera for all your cell culture needsWe provide a simplified three-tiered offering—Gibco™ Value FBS, Premium FBS, and Specialty FBS—where each category is clearly delineated by relevant performance markers and testing levels to help ensure you can confidently select the right serum for your research.Choose the right sera for your specific needs, from basic research to specialty assays. Whether you need sera with the least viral risk, the lowest endotoxin levels, or sera qualified for specialty applications and assays, Gibco products offer you superior value and the clearest choice.Value FBSGibco Value FBS is ideal for standard research applications with up to 50 qualityspecification tests that include 9 CFR virus testing, as well as testing for endotoxinsand performance. Our Value FBS is manufactured using triple 0.1 µm filtration.Testing is performed.* Modified virus testing; see CoA for virus testing.** FBS manufactured in Brazil for Brazil is subjected to double 0.1 µm filtration, not triple (Cat. Nos. 12657011 and 12657029).† If manufactured in the United Kingdom (UK), FBS receives Title 9 of the Code of Federal Regulations (9 CFR) testing, excluding rabies virus and bluetongue virus, which are tested via European Medicines Agency (EMA).Premium FBSChoose Gibco Premium FBS for the lowest risk of bovine spongiform encephalopathy (BSE) and lower viral risk. Our Premium FBS meets USP/EP guidelines with up to 96 harmonized quality specification tests, including European Medicines Agency (EMA) virus testing (selected lots), USP/EP mycoplasma, endotoxin, performance, biochemical/hormonal profiling, and Oritain ™ fingerprinting technology. The serum is manufactured using triple 0.1Testing is performed.• Gamma-irradiated Premium FBS is available upon request.Did you know?9 CFR virus testing: Virus panel testing according to Code of Federal Regulations, (CFR), Title 9, Part 113.53(c) [113.46, 113.47]. Detected by fluorescent antibody.Biochemical hormonal profiling: Quantification of biochemical and hormonal (estradiol, insulin, progesterone, testosterone, and thyroxine) profiling that may have an impact on cell culture.EMA virus testing: Virus panel testing according to EMA/CHMP/BWP/457920/2012 Part 7.3.1 and 7.3.2 and EMEA/CVMP/743/00 Part 4.3.3. Detected by fluorescent antibody.Fingerprinting technology (origin confirmation): A proprietary technology for Gibco sera, to confirm FBS origin and eliminate the potential for counterfeit product.Specialty FBSThese sera are designed for specialty applications and sensitive cell culture, including stem cell research, cancer research, reporter assays, immunoassays, and more.full text of the paper.IT’S ALSO THE MOST TRUSTED SERUM GIBCOIN GLOBAL SCIENTIFIC JOURNALSUsed by 14 of the top 15 pharma companiesUnlike most FBS suppliers, we invest in our own collectors, who obtain the majority of our supply (a by-product of the beef industry) straight fromgovernment-approved facilities with clinically examined healthy animals under veterinary supervision, using only the strictest aseptic collection techniques.At our processing facilities we conduct numerous quality checks, such as testing for hemoglobin levels, to verify that the integrity of the product is maintained.FBS is transferred to a clean room in specially designed stainless steel pipes where itundergoes 0.1 μm triple filtration to minimize biological contaminants.Sterile-filtered serum is immediately and aseptically bottled and undergoesvirus/quality testing before clearing QC.OFFERS THE HIGHEST LEVEL OF TRACEABILITY AND QUALITY MINIMIZED RISK OF CONTAMINATION OF FINAL PRODUCTBlood collectionSterile filtration and processing DispensingRaw serum conversionGibco FBSVERTICALL Y INTEGRATED FINISH-AT-SOURCE MANUFACTURING PROCESSAll products may not be available in all regions due to importation regulations. Contact your sales representative regarding product availability in your country.For Research Use or Further Manufacturing Use Only. Serum and blood proteins are not for direct administrationinto humans or animals . © 2018, 2022 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property L earn more at /fbsGlobal, vertically integrated supply chain for continuity of supply and risk mitigationcGMP–ISO 13485 and/or ISO 9001 facilitiesDifferentiated workflow solutions, fromspecialty serum to innovative packaging like the aliquot-free One Shot FBS 50 mL bottleFBS “fingerprinting” technology—first FBS supplier to develop origin reassuranceiMATCH technology—multiparametric matching tool minimizes lot variation and reduces the need for testingCertified for traceability by the ISIA since 2014Better together—maximize reproducibilityby pairing Gibco FBS and media with Thermo Scientific™Nunc™plastics7 reasons to buy Gibco FBS right nowReferences1. Graham FL et al. (1977) Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol 36(1):59–74.2. Martin G (1981) Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci USA 78(12):7634–7638.3. Wilmut I et al. (1997) Viable offspring derived from fetal and adult mammalian cells. Nature 385(6619):810–813.4. Mali P et al. (2013) RNA-guided human genome engineering via Cas9. Science 339(6121):823–826.。

类号分 级密国际进类号十分（UDC ）破骨细胞功能相关分子CTSK 与ClC-7的临床及基础研究题题（名和副名）薛 洋（作者姓名） 导教师指姓名 毛天球 教授（主任医师）导教师单指位 第四军医大学口腔医院颌面外科请学级别申位 博士专业称名 口腔临床医学(颌面外科学) 论文提交日期 2011.05 辩答日期 2011.05 论时间文起止 2009年04月至2011年04月学单位授予位第四军医大学独创性声明秉承学校严谨的学风与优良的科学道德，本人声明所呈交的论文是我个人在导师指导下进行的研究工作及取得的研究成果。

尽我所知，除了文中特别加以标注和致谢的地方外，论文中不包含其他人已经发表或撰写过的研究成果，不包含本人或他人已申请学位或其他用途使用过的成果。

与我一同工作的同志对本研究所做的任何贡献均已在论文中作了明确的说明并表示了致谢。

申请学位论文与资料若有不实之处，本人承担一切相关责任。

论文作者签名：日期：保护知识产权声明本人完全了解第四军医大学有关保护知识产权的规定，即：研究生在校攻读学位期间论文工作的知识产权单位属第四军医大学。

本人保证毕业离校后，发表论文或使用论文工作成果时署名单位仍然为第四军医大学。

学校可以公布论文的全部或部分内容（含电子版，保密内容除外），可以采用影印，缩印或其他复制手段保存论文。

学校有权允许论文被查阅和借阅，并在校园网上提供论文内容的浏览和下载服务。

论文作者签名：导师签名：日期：破骨细胞功能相关分子CTSK与ClC-7的临床及基础研究研究生：薛 洋学科专业：口腔临床医学（颌面外科学）所在单位：第四军医大学口腔医院颌面外科导师：毛天球 教 授（主任医师）辅导教师：段小红 副教授（副主任医师）关键词：破骨细胞；组织蛋白酶K；致密性成骨不全；氯离子通道7；骨硬化症；分子诊断；牙齿中国人民解放军第四军医大学2011年5月目录缩略语表 (1)中文摘要 (3)英文摘要 (7)前言 (11)文献回顾 (12)正文 (21)第一部分CTSK基因突变所致致密性成骨不全的临床和基础研究 (21)一、159例致密性成骨不全患者的文献回顾性研究 (21)1 资料及方法 (21)2 结果 (22)3 讨论 (29)二、致密性成骨不全的个案研究 (30)1 病例简介 (30)2 临床检查 (30)3 影像学检查及其他辅助检查 (32)4 颌骨骨髓炎的治疗 (35)5 讨论 (37)三、CTSK基因突变分析和家系研究 (40)1 材料及方法 (40)2 结果 (43)3 讨论 (45)四、CTSK参与牙齿发育的基础研究 (47)1 材料及方法 (47)2 结果 (48)3 讨论 (49)五、CTSK与T细胞免疫功能的研究 (52)1 材料及方法 (52)2 结果 (53)3讨论 (55)第二部分CLCN7基因突变所致骨硬化症的临床和基础研究 (57)一、IARO的个案研究 (57)1 病例简介 (57)2 临床检查 (57)3 辅助检查 (59)4 染色体核型分析 (61)5 治疗 (61)6 讨论 (63)二、ADOⅡ的个案研究 (64)1 病例简介 (64)2 临床检查 (64)3 辅助检查 (64)4 临床免疫学检测 (66)5 讨论 (66)三、CLCN7基因突变分析 (68)1 材料及方法 (68)2 结果 (68)3 讨论 (70)第三部分CTSK和ClC-7致病机理的比较分析 (73)一、诊断及鉴别诊断 (73)二、CTSK和ClC-7的致病机理 (75)小结 (79)参考文献 (81)个人简历和研究成果 (104)致谢 (107)缩略语表缩略词英文全称中文全称ADOⅡAutosomal dominant osteopetrosis typeⅡ常染色体显性遗传性骨硬化症Ⅱ型ARO Autosomal recessive osteopetrosis 常染色体隐性遗传性骨硬化症CAⅡCarbonic anhydraseⅡ碳酸酐酶ⅡCCD Cleidocranial dysostosis 颅骨锁骨发育不全ClCs V oltage-gated chloride channels 电压门控氯离子通道家族ClC-7 V oltage-gated chloride channel 7 电压门控氯离子通道7CLC-7 ClC-7 protein ClC-7蛋白CLCN7 Human ClC-7 gene 人ClC-7基因Clcn7 Mouse ClC-7 gene 小鼠ClC-7基因CTSK Cathepsin K 组织蛋白酶KESH Expansile skeletal hyperphosphatasia 广泛性骨骼性高磷酸酯酶血症FEO Familial expansile osteolysis 家族性广泛性骨溶解症HGMD Human gene mutation database 人类基因突变数据库IARO Intermediate autosomal recessiveosteopetrosis 中间型常染色体隐性遗传性骨硬化症JPD Juvenile Paget’s disease 青少年Paget病MMP-9 Matrix metallo proteinase 9 金属基质蛋白酶9-1-缩略词英文全称中文全称OPG Osteoprotegerin 骨保护素骨硬化症相关跨膜蛋白1 OSTM1 Osteopetrosis associatedtransmembrane protein 1RANK Receptor activator of nuclear factor κB核因子κB受体活化因子核因子κB受体活化因子配基RANKL Receptor activator of nuclear factor κBligandPCR Polymerase chain reaction 聚合酶链反应PDB Paget’s disease of bone Paget骨病SNPs Single-nucleotide polymorphisms 单核苷酸多态性位点TCIRG1 T-cell immune regulator 1 T细胞免疫调节剂1（V-ATP酶编码基因）TGP Total glucosides of white peony 白芍总苷Th Helper T cell 辅助性T细胞Th17 T helper 17 辅助性T细胞17Ts Suppressor T cell 抑制性T细胞TRACP Tartrate resistant acid phosphatase 抗酒石酸酸性磷酸酶-2-破骨细胞功能相关分子CTSK与ClC-7的临床及基础研究博士研究生 ：薛洋导 师 ：毛天球 教授第四军医大学口腔医院颌面外科，西安，710032中文摘要骨改建是维持骨骼质量、强度以及钙盐平衡必不可少的过程，是破骨细胞介导的骨吸收过程和成骨细胞介导的骨形成过程协同作用的结果。

泛素特异性蛋白酶7（USP7）在非小细胞肺癌中的研究进展发布时间：2021-11-17T03:43:04.112Z 来源：《健康世界》2021年19期作者：李芝瑜1，何敏诗2，鲁蓝艺2，吴金晓2，郑锦花2[导读] 泛素特异性蛋白酶7 (ubiquitin-specific protease，USP7) 是一种去泛素化酶，可调节众多蛋白的活性，包括肿瘤抑制因子，DNA修复蛋白，免疫应答，病毒蛋白和表观遗传调节剂李芝瑜1，何敏诗2，鲁蓝艺2，吴金晓2，郑锦花2桂林医学院，桂林市 541199；2.桂林医学院附属医院，桂林市 541001摘要：泛素特异性蛋白酶7 (ubiquitin-specific protease，USP7) 是一种去泛素化酶，可调节众多蛋白的活性，包括肿瘤抑制因子，DNA 修复蛋白，免疫应答，病毒蛋白和表观遗传调节剂。

USP7通过去泛素化作用，调控蛋白质的表达，参与肿瘤的多种信号通路的调节，促进肿瘤的发生和发展，是当前癌症治疗研究中人们广泛关注的几种去泛素化酶之一。

本篇综述，总结了对 USP7 研究的进展，重点关注其在非小细胞肺癌中的作用。

关键词：USP7；非小细胞肺癌Research progress of ubiquitin-specific protease 7 (USP7) in non-small cell lung cancerZhiYu Li1，MinShi He2，LanYi Lu2，JinXiao Wu2，JinHua Zheng2（1.Guangxi Key Laboratory of Tumor Immunity and Microenvironment Regulation, Guilin Medical College, 541000, China; 2. Affiliated Hospital of Guilin Medical College, 541000, China）Abstract: Ubiquitin-specific protease 7 (USP7) is a deubiquitinating enzyme that can regulate the activity of many proteins, including tumor suppressor factors, DNA repair proteins, immune responses, viral proteins, and epigenetics Modifier. USP7 regulates protein expression through deubiquitination, participates in the regulation of multiple signal pathways in tumors, and promotes the occurrence and development of tumors. It is one of several deubiquitinating enzymes that have been widely concerned in current cancer treatment research. This review summarizes the research progress of USP7, focusing on its role in non-small cell lung cancer.Keywords: USP7; non-small cell lung cancer现如今肺癌已经成为全球发病率和死亡率最高的恶性肿瘤之一，5年生存率＜16%[1]，已成为影响生存率的第一大致死原因，尽管广大医务工作者为应对这种恶性疾病付出了数十年的努力，但肺癌的预后仍然不乐观，尤其是晚期非小细胞肺癌 (NSCLC)。

第 2 章细胞工程第 1 节植物细胞工程01 植物细胞工程的基本技术1.P36从社会中来：其芽葺葺,其叶青青,犹绿衣郎,挺节独立,可敬可幕。

迨夫花开，凝晴滚露，万态千妍，薰风自来，四坐芬郁，岂非入兰室乎!岂非有国香乎!”这是我国历史上第一部兰谱一-《金漳兰谱》(宋.赵时庚)中对兰花的一段描述。

从古至今，我国人民都把兰花看作高洁、典雅的象征，很多人喜欢养兰花。

但是，兰花种子通常发育不全，在自然条件下萌发率极低;传统分株繁殖的方法又存在繁殖周期长、繁殖率低等问题，如果靠自然繁殖，兰花的价格可想而知了。

如何能让名贵的兰花大量、快速地繁殖，从而走入寻常百姓家呢?提示:利用植物组织培养技术可以大量、快速地培育兰花。

兰花是观赏植物中最常见的一类依靠植物组织培养繁育种苗的植物，其组培苗的数量约占观赏植物组培苗总量的40%.2.P36探究·实践：菊花的组织培养(1)接种3-4d后，检查外植体的生长情况，统计有多少外植体被污染，有多少能正常生长，并分析它们被污染的原因。

根了吗?用于植物组织培养的培养基同样适合某些微生物的生长，如一些细菌、真菌等的生长。

培养物一旦受到微生物的污染，就会导致实验前功尽弃，因此，要进行严格的无菌操作。

导致外植体被污染的原因可能有：培养基、接种工具灭菌不彻底；外植体消毒不彻底；操作过程不符合无菌操作要求等。

(2)你培养出愈伤组织了吗?如果培养出来了，从刚接种的外植体到长出愈伤组织经历了多少天?这些愈伤组织进一步分化出芽和提示：观察实验结果，看看是否培养出了愈伤组织，记录多长时间长出了愈伤组织。

从刚接种的外植体到长出愈伤组织一般需要2周左右的时间。

统计更换培养基后愈伤组织进一步分化成芽和根的比例和时间。

(3)观察外植体的分化情况，填好结果记录表，并及时分析结果。

提示：做好统计和对照，填好结果记录表，培养严谨的科学态度。

从实验的第一步开始就要做好实验记录，可以分组配制不同的培养基，如诱导愈伤组织的培养基、诱导生芽的培养基等，还可以进行不同配方的比较。

环磷酸腺苷（cAMP）的营养保健功能成分实验研究刘孟军河北农业大学科技处中国枣研究中心一．认识cAMPcAMP是一种蛋白激酶制活剂，它的中文名字叫环磷酸腺苷，它与环磷酸鸟苷（cGMP）并称为环核苷酸，二者的比例约为50：1，是受神经内分泌系统控制的下属单位，是中枢神经细胞的“第二信使”，作为肽类激素、儿茶酚胺和前列腺等激素的第二信使发挥生物效应，对中枢神经系统活动起重要调节作用；环核苷酸可以调节基因的活动，促进mRNA基因的转录，影响酶的合成，起到代谢调节的作用，与人体的生命活动、疾病的发生、发展和康复、病理生理的变化都息息相关。

二．cAMP对人体的重要性cAMP可以促进人体的三大物质代谢，在细胞水平的调节上有非常重要的作用。

它可以促进脑细胞的新陈代谢及功能修复，缓解脑细胞疲劳，延缓脑细胞的衰老，对增强记忆力有非常良好的作用。

在2000年，有一位叫做坎德尔的美国科学家发现记忆的形成机制而获得诺贝尔生理及医学奖，他发现无论是短时记忆还是长期记忆都与cAMP密切相关，蛋白质磷酸化对记忆形成中分子机制的作用，在坎德尔获奖的理论中是极为重要的一环。

短期记忆由较弱的刺激形成。

神经递质促进cAMP制造→cAMP再活化蛋白质活化酶A （PKA）→PKA再使特定离子管道（如钾离子管道）蛋白质磷酸化→导致神经递质在突出的释放增加可，就形成短期的记忆。

较强和较久的刺激即形成长期记忆。

因此我们可以看出cAMP在整个记忆形成过程中所起到的重要作用，它的缺乏与不足将严重影响后续过程，导致记忆力低下！医学研究证明至少有40多种疾病与环核苷酸的代谢有关，人体内缺乏环磷酸腺苷（cAMP）即cAMP/cGMP值下降，会导致恶性肿瘤、癌症、失眠健忘、贫血、心血管病等众多疾病的发生。

三．cAMP研究与发展1957年，美国科学家萨瑟蓝德发现并报道了cAMP这种物质的存在及其相应的作用机理，他也因此荣膺1971年的诺贝尔生理及医学奖。

自从cAMP被发现以后，全世界上千所实验室都在对它进行研究。

[收稿日期]㊀2020-11-27[修回日期]㊀2021-02-11[基金项目]㊀湖南省教育厅科学研究重点项目(19A429)[作者简介]㊀李娟,硕士研究生,研究方向为放射医学,E-mail 为lijuan9508@㊂通信作者黄波,博士,硕士研究生导师,副教授,研究方向为放射医学,E-mail 为huangbo0930@㊂㊃基础医学㊃DOI :10.15972/ki.43-1509/r.2021.03.011NU7026对人肝癌细胞HepG2生物学行为的影响李娟,颜宇龙,万丹婷,李蕊,周美艳,周洁,黄波(南华大学公共卫生学院,湖南省衡阳市421001)[关键词]㊀NU7026;㊀肝癌;㊀HepG2细胞;㊀增殖;㊀迁移[摘㊀要]㊀目的㊀探讨NU7026对HepG2细胞的影响,为治疗肝癌提供理论基础㊂方法㊀用NU7026作用HepG2细胞,将实验分为空白组㊁对照组和NU7026组㊂CCK8实验检测NU7026对肝癌HepG2细胞存活率的影响㊂生长曲线实验和克隆实验检测HepG2细胞增殖能力,划痕实验检测HepG2细胞迁移能力,流式细胞术检测HepG2细胞凋亡和周期㊂结果㊀与0μmol /L 组和DMSO 组比较,15μmol /L 和20μmol /L NU7026作用于HepG2细胞,细胞存活率明显下降(P <0.05);与DMSO 组相比,10μmol /L NU7026作用于HepG2细胞24㊁48㊁72h 后,细胞存活率下降(P <0.05);10μmol /L NU7026作用HepG2细胞,细胞增殖能力和迁移能力下降(P <0.05),细胞凋亡率增加(P <0.05),细胞周期无明显改变㊂结论㊀NU7026能抑制HepG2细胞增殖能力和迁移能力可能与促进细胞凋亡有关㊂[中图分类号]㊀R735.7[文献标识码]㊀AEffect of NU 7026on the biological behavior of human hepatocarcinoma cell HepG 2LI Juan,YAN Yulong,WAN Danting,LI Rui,ZHOU Meiyan,ZHOU Jie,HUANG Bo (School of Public Health ,University of South China ,Hengyang ,Hunan 421001,China )[KEY WORDS ]㊀NU7026;㊀liver cancer;㊀HepG2cell;㊀proliferation;㊀migration [ABSTRACT ]㊀㊀Aim ㊀To explore the effect of NU7026on HepG2cells,and provide a theoretical basis for the treat-ment of liver cancer.㊀㊀Methods ㊀HepG2cells were treated with NU7026,and the experiment was divided into the blank group,the control group and the NU7026group.㊀The CCK8assay was used to detect the effect of NU7026on the survival rate of HepG2cells.㊀The growth curve assay and clone formation assay were used to detect the proliferation ability of HepG2cells,and the scratch assay was used to detect the migration ability of HepG2cells.㊀Finally the cellular apopto-sis and cell cycle were tested by flow cytometry.㊀㊀Results ㊀Compared with the 0μmol /L group and the DMSO group,after 15μmol /L and 20μmol /L NU7026treated on HepG2cells,the cell survival rate was significantly decreased (P <0.05);Compared with the DMSO group,after treating HepG2cells with 10μmol /L NU7026for 24,48,and 72hours,the cell survival rate was significantly decreased (P <0.05);After 10μmol /L NU7026treated HepG2cells,the cell prolif-eration and migration ability decreased (P <0.05),the apoptosis rate was significantly increased (P <0.05),but the cell cycle did not change significantly.㊀㊀Conclusion ㊀NU7026can inhibit the proliferation and migration of HepG2cells,which may be related to the promotion of cell apoptosis.㊀㊀肝癌是世界上最常见的恶性肿瘤之一,2015年中国肝癌发病人数为37.0万人,发病率为26.92/10万,死亡人数32.6万,死亡率23.72/10万[1]㊂原发性肝癌发病隐匿,病程短,发展迅速,当前主要治疗手段有手术切除㊁放射治疗㊁药物治疗㊁肝移植等㊂但肝癌易复发㊁易转移,预后差,且对放化疗具有抵抗性,寻求治疗肝癌的药物具有重要意义㊂原发性肝癌中最常见的肝癌组织类型是肝细胞癌[2]㊂本文探讨了NU7026抑制DNA-PKcs 对肝癌细胞的增殖㊁迁移能力的影响,为治疗肝癌提供理论依据㊂1㊀材料和方法1.1㊀材料㊀㊀肝癌细胞(HepG2)由军事科学医学院周平坤研究员惠赠㊂HepG2细胞在含10%胎牛血清(杭州四季青公司)的DMEM培养基(Gibco),37ħ㊁5%CO2培养箱中培养㊂NU7026(LY293646,C17H15NO3)购于MCE公司,纯度为99.95%㊂细胞凋亡试剂盒㊁细胞周期试剂盒购于南京凯基公司㊂1.2㊀CCK8实验取处于指数生长期的HepG2细胞,用含10%胎牛血清的培养液配成单个细胞悬液接种于96孔板中,加入适量的细胞悬液(约7000~8000个/孔),待细胞贴壁后,分别用5㊁10㊁15㊁20μmol/L NU7026进行处理,设3个复孔,继续培养24㊁48㊁72h,每孔加入10μL CCK8溶液,孵育1h,用酶联免疫检测仪测定450nm波长处的光密度值(OD),计算细胞存活率㊂细胞存活率(%)=(实验组OD值-空白孔OD值)/(空白组OD值-空白孔OD值)ˑ100%㊂1.3㊀生长曲线实验按2ˑ104个/孔细胞接种于6孔细胞培养板, 10μmol/L NU7026处理细胞每组设3个复孔,每隔24h计数1次,连续计数3天,以时间为横坐标,细胞数为纵坐标绘制生长曲线图㊂1.4㊀克隆实验将细胞接种于6cm细胞培养皿,10μmol/L NU7026处理细胞,10天后弃掉培养基,PBS洗2遍,加入固定液3mL,固定30min;再加入3mL Gi-emsa染色30min,最后用流水缓慢冲洗,晾干㊂计数含50个细胞以上的克隆数,克隆形成率(%)=克隆数/[实验组接种细胞数ˑ(对照组克隆数/对照组接种细胞数)]ˑ100%㊂1.5㊀划痕实验将细胞接种在6孔细胞培养板中,细胞贴壁后用200μL无菌的枪头尖端在每孔相同位置做线性划痕,再用PBS轻轻冲洗2~3遍,10μmol/L NU7026处理细胞,分别在0㊁24㊁48㊁72h后于显微镜下拍照观察划痕,采用Image J软件观察计算细胞划痕面积,面积愈合率(%)=(0h划痕面积-培养后划痕面积)/0h划痕面积ˑ100%㊂1.6㊀细胞凋亡与细胞周期试验将细胞接种于6孔细胞培养板,10μmol/L NU7026处理细胞,按照说明书步骤进行,1h内用流式细胞仪检测细胞凋亡情况㊂取适量HepG2细胞接种于6cm细胞培养皿中,用10μmol/LNU7026处理0㊁4㊁8㊁12㊁24h后收样㊂按照说明书步骤进行,用流式细胞仪检测细胞周期㊂1.7㊀统计学分析采用SPSS18.0进行统计分析,数据以xʃs表示,多组间用单因素方差分析,P<0.05为差异有显著性㊂2㊀结㊀果2.1㊀不同浓度NU7026对HepG2细胞活性的影响随着NU7026浓度的增加,HepG2细胞存活率下降㊂与0μmol/L组和DMSO组比较,15μmol/L 和20μmol/L NU7026作用HepG2细胞后,细胞存活率下降(P<0.05;图1);10μmol/L NU7026作用HepG2细胞24㊁48㊁72h后,与DMSO组细胞存活率比较,差异有显著性(P<0.05),与0μmol/L 组细胞存活率比较,差异无显著性(P>0.05);查阅文献[3]及根据前期研究[4],本研究中10μmol/L NU7026对HepG2细胞没有明显的毒性作用,故选择10μmol/L NU7026进行后续实验㊂将实验分为空白组㊁对照组(0.1%DMSO)和NU7026组(10μmol/L NU7026)㊂图1㊀CCK8检测不同浓度NU7026对HepG2细胞活性的影响a为P<0.05,与0μmol/L组比较;b为P<0.05,与DMSO组比较㊂2.2㊀各组HepG2细胞增殖能力比较NU7026作用HepG2细胞72h后,NU7026组细胞数明显低于对照组(P<0.05;图2);NU7026组细胞克隆数少于对照组,克隆形成率明显降低(P< 0.05;图2)㊂图2㊀各组HepG2细胞增殖能力的比较A 为培养10天后克隆实验细胞分布图(Giemsa 染色)和柱状图;B 为细胞生长曲线图㊂a 为P <0.05,与空白组比较;b 为P <0.05,与对照组比较㊂2.3㊀各组HepG2细胞迁移能力NU7026作用细胞24h 后,与对照组相比,面积愈合率无明显差异(P >0.05);NU7026作用细胞48h后,划痕面积比对照组稍大,面积愈合率无明显差异(P >0.05);但在作用72h 后,NU7026组划痕面积大于对照组,面积愈合率明显降低(P <0.05;图3)㊂图3㊀各组HepG2细胞迁移能力比较A 为细胞划痕实验迁移(200ˑ);B 为细胞划痕愈合率折线图㊂a 为P <0.05,与空白组比较;b 为P <0.05,与对照组比较㊂2.4㊀各组HepG2细胞的凋亡与周期NU7026作用HepG2细胞24㊁48㊁72h 后,细胞凋亡率增加(P <0.05;图4)㊂随着NU7026作用时间增加,HepG2细胞凋亡率逐渐增加㊂NU7026作用HepG2细胞12h 后,出现了轻微的G2/M 阻滞现象,但细胞周期未出现明显改变(图5)㊂3㊀讨㊀论肝癌在全球恶性肿瘤发病谱中排第六,其中中国肝癌新发病例数将近占全球发病的一半[5]㊂DNA-PKcs 是一种相对分子质量约为470kDa 的丝氨酸/苏氨酸蛋白激酶,与共济失调毛细血管扩张症基因(Ataxia telangiectasia mutated,ATM)㊁RAD3相关蛋白(Ataxia telangiectasiaand Rad3-related protein,ATR)同属于磷酸酰肌醇3-激酶相关蛋白激酶(PIKK)家族㊂DNA-PKcs 和ATM 在DNA 双链断裂修复中起着重要的作用,ATR 主要参与DNA 单链断裂修复㊂DNA-PKcs 不仅参与淋巴细胞的V(D)J 重组和类别转换重组,保护染色体末端,而且DNA-PKcs 响应胰岛素信号传导,促进肝脏中碳水化合物转化为脂肪酸[6-7]㊂DNA-PKcs 抑制剂有渥曼青霉素㊁NU7441㊁NU7026㊁IC 化合物等㊂NU7026对DNA-PKcs 抑制性特异度高,能有效抑制DNA-PKcs Ser2056磷酸化[4]㊂本研究通过生长曲线实验和克隆实验检测图4㊀各组HepG2细胞凋亡比较A 为细胞凋亡图;B 为细胞凋亡率柱状图㊂a 为P <0.05,与空白组比较;b 为P <0.05,与对照组比较㊂图5㊀各组HepG2细胞周期的比较A 为流式细胞术检测细胞周期图;B 为细胞周期占比图㊂细胞增殖能力,表明NU7026抑制DNA-PKcs 将降低HepG2细胞增殖能力,与其他研究结果相一致㊂用siRNA 介导肝癌HepG2细胞中DNA-PKcs 沉默,细胞增殖速度减慢,且高表达DNA-PKcs 可通过AKT /GSK3/c-myc 信号通路调节肝癌细胞增殖[8]㊂同样用siDNA-PKcs 转染肝癌耐药细胞Bel-7402/5-Fu 后,细胞增殖能力降低,可能抑制DNA-PKcs 后胸腺嘧啶合成酶(TS)表达下降,从而抑制DNA 合成,细胞增殖生长变慢[9]㊂但也有研究表明NU7441抑制DNA-PKcs 促进肺成纤维细胞中SSEA4+间充质祖细胞增殖[10]㊂沉默DNA-PKcs 不仅抑制骨肉瘤MG-63细胞增殖,而且也抑制细胞迁移和侵袭[11]㊂本文结果显示抑制DNA-PKcs 能降低HepG2细胞迁移能力㊂有研究表明将沉默DNA-PKcs 的HepG2细胞移植到裸鼠中,其肿瘤生成率明显降低[8]㊂DNA-PKcs通过RhoA/ROCk2通路调节基质金属蛋白酶-2和磷酸化肌球蛋白轻链2表达,促进体内外骨肉瘤细胞迁移和侵袭[12]㊂也有研究发现ITGA5和SDC4基因与DNA-PKcs可能对细胞迁移侵袭运动具有互相调节作用[13]㊂本研究用NU7026抑制DNA-PKcs24㊁48㊁72h, HepG2细胞凋亡增加,与其他研究结果一致㊂有研究表明沉默DNA-PKcs后,通过线粒体凋亡途径促进肝癌耐药细胞Bel-7402/5-Fu凋亡[14]㊂也有研究表明沉默DNA-PKcs后,有丝分裂细胞纺锤体异常,胞质分裂失败,有丝分裂延长等,最终导致细胞有丝分裂灾变而凋亡[15]㊂用TDR将HeLa细胞同步化至G1期后释放,沉默DNA-PKcs的HeLa细胞在6h出现明显的G2/M期阻滞,表明抑制DNA-PKcs会引起G2/M期阻滞[16]㊂用nocodazole同步化细胞后释放,抑制HeLa细胞DNA-PKcs,H3-pS10阳性细胞增加,表明细胞阻滞在有丝分裂期[17]㊂也有研究表明沉默MG-63细胞DNA-PKcs48h,细胞周期无明显改变[11]㊂在本研究中NU7026抑制DNA-PKcs对HepG2细胞周期无明显改变,可能是因为细胞DNA 损伤不严重,DNA修复时间较短,细胞能较快地通过周期检查点,故未出现明显G2/M期阻滞㊂综上所述,NU7026能抑制HepG2细胞增殖能力和迁移能力,其机制可能与促进细胞凋亡有关㊂[参考文献][1]郑荣寿,孙可欣,张思维,等.2015年中国恶性肿瘤流行情况分析[J].中华肿瘤杂志,2019,41(1):19-28.[2]龙晓兰,张万勇,吴勇军,等.Cofilin-1与肝癌细胞迁移侵袭能力的相关性研究[J].中南医学科学杂志, 2019,47(6):571-575.[3]AMREIN L,LOIGNON M,GOULET A C,et al.Chloram-bucil cytotoxicity in malignant B lymphocytes is synergisti-cally increased by2-(morpholin-4-yl)-benzo[h]chomen-4-one(NU7026)-mediated inhibition of DNA double-strand break repair via inhibition of DNA-dependent protein kinase [J].J Pharmacol Exp Ther,2007,321(3):848-855.[4]黄波,龙颖,唐艳,等.NU7026对肝癌细胞放射增敏作用及机制研究[J].辐射研究与辐射工艺学报,2013, 31(6):11-15.[5]安澜,曾红梅,郑荣寿,等.2015年中国肝癌流行情况分析[J].中华肿瘤杂志,2019,41(10):721-727.[6]SOULAS-SPRAUEL P,RIVERA-MUNOZ P,MALIVERT L,et al.V(D)J and immunoglobulin class switch recom-binations:a paradigm to study the regulation of DNA end-joining[J].Oncogene,2007,26(56):7780-7791. [7]CHUNG J H.The role of DNA-PK in aging and energy me-tabolism[J].FEBS J,2018,285(11):1959-1972.[8]黄越承,周平坤,蔡建明,等.肝癌及不同发育阶段小鼠肝组织中DNA-PKcs的表达及其对细胞增殖作用的研究[J].第二军医大学学报,2009,30(11): 1217-1220.[9]李大玉,余春波,束波,等.DNA-PKcs影响肝癌耐药细胞Bel-7402/5-Fu增殖及机制的研究[J].贵州医药, 2016,40(2):131-134.[10]HABIEL D M,HOHMANN M S,ESPINDOLA M S,et al.DNA-PKcs modulates progenitor cell proliferation and fi-broblast senescence in idiopathic pulmonary fibrosis[J].BMC Pulm Med,2019,19(1):165.[11]JIN P Y,LU H J,TANG Y,et al.The effect of DNA-PKcs gene silencing on proliferation,migration,invasion and apoptosis,and in vivo tumorigenicity of human osteo-sarcoma MG-63cells[J].Biomed Pharmacother,2017, 96:1324-1334.[12]李卡.DNA-PKcs对CD133阳性骨肉瘤细胞顺铂耐药性和骨肉瘤转移的作用及机制研究[D].济南:山东大学,2017:1-190.[13]宋志全.电离辐射对DNA损伤修复蛋白DNA-PKcs及其结合RNA作用的研究[D].北京:北京市军事科学院,2019:1-72.[14]余春波,李大玉,范芳,等.线粒体凋亡途径在DNA-PKcs抑制肝癌耐药细胞BEL7402/5-Fu凋亡中的作用研究[J].遵义医学院学报,2019,42(3):260-264.[15]SHANG Z F,HUANG B,XU Q Z,et al.Inactivation ofDNA-dependent protein kinase leads to spindle disruption and mitotic catastrophe with attenuated checkpoint protein 2phosphorylation in response to DNA damage[J].Cancer Res,2010,70(9):3657-3666. [16]AN J,HUANG Y C,XU Q Z,et al.DNA-PKcs plays adominant role in the regulation of H2AX phosphorylation in response to DNA damage and cell cycle progression [J].BMC Mol Biol,2010,11:18.[17]HUANG B,SHANG Z F,LI B,et al.DNA-PKcs associ-ates with PLK1and is involved in proper chromosome seg-regation and cytokinesis[J].J Cell Biochem,2014,115(6):1077-1088.(此文编辑㊀蒋湘莲)。

Application Note ZEISS Celldiscoverer 7High quality LSM imaging with autocorrection objectivesusing plastic labware.Figure 1 Overview of ZEISS Celldiscoverer 7 objectives. In combination with the 3-position magnification changer, a total of 12 magnifications with individual numerical apertures can be used.ZEISS Celldiscoverer 7 – an automated live cell imaging system designed to make complex microscopy simple – harbors many automation functions such as automatic sample recognition, measurement of bottom material and bottom thickness, an auto-calibration routine for micro-well-plates, an auto-immersion water objective and a variety of autofocus options to name but a few. All these features were assembled in this machine to enable easy execution of sophisticated experiments for various types of samples and sample carriers.Authors: S andra Orthaus-Müller, Volker Döring, Frank Vogler, Harald SchadwinkelCarl Zeiss Microscopy GmbH, GermanyDate: February 2021IntroductionThe optical concept of this boxed microscope consists of two elements: Up to four objectives are situated in a 4-position objective turret underneath the sample and a 3-position magnification changer enables fast and easy switching of the total magnification of eacho bjective. In this way, 12 different com -binations are available allowing a range from 2.5× up to 100× magnification with high NA (see figure 1). In addition, all objectives in the Cell d iscoverer 7 feature apochromatic correction to enable artefact-free multicolor imaging.To ensure optimal environmental condi-tions for live cell experiments, the front lenses of the objectives as well as the bottom and lid of the sample chamber are heated to the desired temperature.The three objectives with the highest numerical apertures include a motorized autocorrection ring. In combination with the information regarding the sample carrier bottom material (including its refractive index and thickness – both are determined automatically upon sample loading), optimal image acquisition is guaranteed by adjusting the objective to the optimal autocorrection ring position (see figure 2). The automatic bottom material detection corrects for any refractive index mismatch, while the automatic bottom thickness mea-surement is used to avoid spherical and chromatic aberrations. For an in-depth review of the influence of objective and sample features as well as the advantage of autocorrection optics please refer to the info box.Figure 2 Autocorrection objectives. ZEISSCelldiscoverer 7 objectives include an autocorrection ring to automatically adopt to bottom material and thickness. This way an optimal image quality is ensuredfor all sample carriers in every acquisition.The introduction of the LSM 900 including the Airyscan 2 transforms ZEISS Celldiscoverer 7 into the first fully automated confocal imaging system on the market. The High Sensitivity (HS) mode of the Airyscan 2 detector enables very gentle imaging because the amount of excitation light necessary for image acquisition is greatly reduced. At the same time, the signal-to-noise ratio and hence the resolution is clearly improved when compared to standard confocal imaging. Applying the Airyscan multiplex 2Y mode using two lines for imaging inone sweep, the acquisition speed is also increased. The three classic features of confocal imaging: speed, resolution and signal-to-noise ratio, must be carefully balanced with respectto one another. Focusing on one aspect automatically means a reduction in the two other parameters. With Airyscan multiplex 2Y mode, all three aspects can now be improved at the same time. This turns ZEISS Celldiscoverer 7 into an extremely sensitive high-resolution point scanner that is ideally suited for long term live-cell imaging experiments especially in combination with 25× water autoimmersion objective with an outstanding numerical aperture of 1.2.Very often, experimental conditions or cost prohibit the use of optical grade glass bottom material with a typical thickness of 170 µm. As an alternative, in many cases rather thick (~1 mm) optically plastic material is used to accommodate demanding cell cultures. In a classic fluorescence microscope this kind of setup would lead to sub-standard imaging results with many aberrations as well as loss of excitation and emission light. Since ZEISS Celldiscoverer 7 automatically adjusts to the vessel bottom (material as well as thickness), the highest image quality is always maintained ensuring that the maximum available information can be gathered from the sample. This is especially true when imaging in LSM mode through optically low-quality material like plastic vessels with thicknesses of 1mm or more. In this particular case, the focused laser beam that is scanned into the sample, as well as the collected emitted light from the sample that needs to pass the pinhole at the conjugated plane, is heavily distorted if no proper correction is performed.Figure 3 Comparison of image quality of A) thin glass bottom (170 µm) and B) thick plastic bottom when the optimal correction is applied. U2OS Cells stained with Hoechst (blue), MitoTracker ™ Red (red), Alexa Fluor ™ 488 Phalloidin (green)Aglass bottomBplastic bottom20 µmFigure 4 Comparison of image quality and different laser powers under A: optimalautocorrection ring settings (1,020 µm). Laser power: DAPI (blue) 2.5 % 405 nm, Mitotracker (magenta) 1.5 % 561 nm; B: non-optimal autocorrection ring settings (210 µm). Laser power: DAPI (blue) 25 % 405 nm, MitoTracker TM Red (magenta) 80 % 561 nm.optimalnon-optimalIn this article the difference in confocal image quality, signal-to-noise ratio (SNR) and excitation power is compared between optimal and incorrect autocorrection ring settings for cells grown on thick plastic (>1000 µm). The findings demonstrate the power of the autocorrection objectives for high-quality confocal imaging using plastic bottom sample carriers.ResultsAs described above, the image quality – or more precisely resolution and SNR – are being influenced by the illumination and detection objective as well as sample features like bottom material and thickness of the sample carrier, refractive index of the medium and imaging depth. If this information is known, the image quality can be improved by reducing spherical and chromatic aberrations with the help of correction optics. ZEISS Cell d iscoverer 7 automatically detects bottom material and thickness upon sample loading. This information is then used to automatically set the autocorrection rings at the objectives to the optimal position to correct for any negative influence on the image quality.The following examples showcase adherent cells grown on different imaging vessels that have either thin glass (~170 µm) or thick plastic (~1000 µm) bottom material – the confocal imaging was performed using the Airyscan 2 detector deploying an automatically corrected 20× objective with a numerical aperture of 0.7.Figure 3 shows the strength of the adaptive optics of ZEISS Celldiscoverer 7. Cells that are either grown on optically well-suited glass of a thickness of 170 µm compared to cells on a thick 1000 µm plastic bottom – a challenging setup for standard confocal fluorescence microscopy. While there is a higher clarity of the cells grown on glass, the plastic harboredcells still showcase an astounding amount of detail due to the optimal adaptation to bottom material and thickness that was automatically applied to the objective.When comparing the image quality between the automatically calculated setting for a vessel bottom thickness of 1020 µm versus an incorrect setting manually overwritten to a thickness of 210 µm, it is apparent that due to the missing optimization the image quality of the latter is severely impacted when compared to the corrected version (see figure 4). As the light is far blurrier due to spherical aberrations and refractive index mis-match, the image appears fuzzy and fine details are no longer visible resulting in a loss of resolution. In addition, aberrations not only manifest themselves when light is detected from the sample but also appear when light is being focused (or in this case rather not properly focused) into to specimen to stimulate fluorescence. This combination of loss of optical quality on the detection-side and reduced excitation efficiency means that excessive amounts of excitation light (10-50-fold increase in this example) are needed to get an image with comparable brightness values. An alternative to increasing the laser power could be an increase in pixel dwell time, however neither option is ideal for live cell imaging experiments as the former very likely induces phototoxic effects while the latter prevents the observation of fast processes as the maximum acquisition speed is significantly reduced.Figure 5 The influence of autocorrection objectives on chromatic and spherical aberrations as well as laser intensities and overall image quality. ZEISS Celldiscoverer 7 automatically measures bottom thickness and adjusts the autocorrection rings to correct for these artefacts. Comparison of 3-dimensional datasets from a DAPI (green) and MitoTracker ™ Red (red) stained U2OS Cell. A: optimal autocorrection ring settings (@1,020 µm bottom thickness; 405 nm = 3.8%; 561 nm = 2.8%). B: non-optimal autocorrection ring settings (@210 µm bottom thickness; 405 nm = 80%; 561 nm = 25%) C: non-optimal autocorrection ring settings (@510 µm bottom thickness; 405 nm = 80%; 561 nm = 50%) D: non-optimal autocorrection ring settings (1,200 µm bottom thickness + 100 µm imaging depth; 405 nm = 20%; 561 nm = 20%). In the autocorrection ring settings, the refractive index of the bottom material is also considered.When looking at 3-dimensional data (figure 5), it is apparent that the z-resolution is drastically impacted if the autocorrection ring is not set properly. When the correct auto-detected and -adjusted setting of 1020 µm thickness is used, the cell appears thin in its z-expansion and both colors align in the same z-plane (figure 5A). One can clearly distinguish the flat lower side of the cell that resides on the plastic surface from the more rounded top side that extends into the medium. Manually changing the autocorrection ring setting to a different (= wrong) value will clearly decreases the image quality – the cell appears to be elongated in the z-direction and the two colors no longer line up in the same z-plane (figure 5B-D).This demonstrates the appearance of spherical and chromatic aberrations if the optics are not properly corrected for bottom thickness. In addition to these aberrations, the laser power needs to be significantly increased to compensate for the improper correction ring settings.A optimal 1,020 μmCnon-optimal 510 μmBnon-optimal 210 μmDnon-optimal 1,200 + 100 μm5 µmSummaryModern long term Live-Cell-Imaging applications that last up toseveral days have a high demand for instrument performancelike environmental control (temperature and CO₂), a stablefocus and gentle illumination parameters to mimic the in vivoconditions of the investigated sample as closely as possible. Allthese features are included in ZEISS Celldiscoverer 7. In addition, autocorrection objectives supplied with the instrument allowfor the use of different types of sample carriers consisting ofdifferent bottom material and thickness. The machine will auto-matically detect and adjust these parameters whenever a newsample is being loaded. This way even optically non-ideal vesselssuch as those with thick plastic bottoms (> 1 mm) can be used togenerate high quality imaging data. The information gatheringcapability is maintained. This even works in confocal mode incombination with the Airyscan 2 detector of the integratedLSM 900 as shown in this article. Distortions like chromaticand spherical aberrations are corrected for ensuring that imagequality parameters like high resolution and signal-to-noise ratioare optimized. In addition, correctly adjusted optics allow forhigh-speed image acquisition and guarantee low phototoxicityfor long-term experiments, as the necessary light dosage forfluorescence imaging is greatly reduced. This, in combinationwith the other features, turn ZEISS Celldiscoverer 7 into an ideallive cell imaging system that is fully automated and enables theexecution of very sophisticated applications.Cover image: Sample courtesy of J. Hartmann and D. Gilmour, EMBL, Heidelberg, GermanyCarl Zeiss Microscopy GmbH 07745 Jena, Germany******************** /celldiscoverer Notallproductsareavailableineverycountry.Useofproductsformedicaldiagnostic,therapeuticortreatmentpurposesmaybelimitedbylocalregulations.ContactyourlocalZEISSrepresentativeformoreinformation.EN_41_13_243|CZ2-221|Design,scopeofdeliveryandtechnicalprogresssubjecttochangewithoutnotice.|©CarlZeissMicroscopyGmbH。