医药学类文献双语版：汉译英

1、背景由于肿瘤细胞抑制凋亡增殖 , 特定凋亡的抑制因素会对于发展新的治 疗策略提供一个合理途径。 Livin 是一种凋亡抑制蛋白家族成员，在多种恶性肿 瘤的表达中具有意义。但是 , 在有关胃癌方面没有可利用的数据。在本研究中 , 我们发现livin 基因在人类胃癌中的表达并调查了介导的shRNA能抑制肺癌细胞中livin 沉默基因的表达，从而促进SGC-7901细胞凋亡。方法一mRN及蛋白质livin基因的表达用逆转录聚合酶链反应技术及西方吸 干化验进行了分析。小干扰 RNA真核表达载体具体到livin基因采用基因重组、 测序核酸。然后用Lipofectamin2000转染进入SGC-7901细

2、胞。逆转录聚合酶链 反应技术和西方吸干化验用来验证的livin基因在SGC-7901细胞中使沉默基因生 效。所得到的稳定的复制品用 G418来筛选。细胞凋亡用应用流式细胞仪(FCM)来 评估。细胞生长状态和5-FU的50%卬制浓度(IC50)和顺铂都由MTTt匕色法来决定。结果一livin mRN和蛋白质的表达检测40例中有19例有胃癌和SGC-7901 细胞。没有 livin 基因表达的是在肿瘤邻近组织和良性胃溃疡病灶。相关发现在 livin 基因的表达和肿瘤的微小分化和淋巴结转移一样 (P < 。 4 个小干扰 RNA 真核表达矢量具体到基因重组的 livin 基因建立。 其中之一

3、,能有效地减少 livin 基因的表达 ,抑制基因不少于 70%(P < 。重组的质粒被提取和转染到胃癌细胞。 G418筛选所得到的稳定的复制品被放大讲究。当livin 基因沉默,胃癌细胞的生殖活动明显低于对照组 (P < 。研究还表明 ,IC50 上的 5-Fu 和顺铂在胃癌细胞的 治疗上是通过shRNA减少以及刺激这些细胞(5-Fu proapoptotic 和顺铂)(P <。结论 livin 基因在胃癌中的过分表达与肿瘤分化与淋巴结转移建立联系 , 建议了治疗胃癌病例分子预后因素之一。 ShRNA可以抑制在SGC-7901细胞中的 livin 基因表达, 诱导细胞凋亡。

4、 Livin 可以作为治疗胃癌凋亡的新目标。1 介绍胃癌是世界上最常见的恶性肿瘤之一。 大多数患者被诊断为这个疾病的阶段 , 在最佳时间的机会错失了手术治愈。 尽管有很大改善 ,但处于晚期胃癌的化疗患者 的总体存活率仍然很低。癌症细胞化疗耐抗性可能导致手术失败。在耐药的原因 中,抑制细胞凋亡的过程会起重要作用。癌细胞常有抗凋亡增长的特征 1,介导其 增加的阻力不同来刺激的细胞凋亡,如DNA损伤、缺氧、营养损失2、3。此外,在 临床实践中细胞凋亡抵抗被认为是肿瘤手术失败的主要原因 ,因此许多化疗药物 和/ 或放射线疗法 都是通过诱导凋亡肿瘤死亡实现的 4。酶抑制剂 (IAPs), 是一种新型的凋

5、亡蛋白抑制基因家族 5,6 ,包括病毒感染 , 化疗药物,生长因子和肿瘤坏死因子-a(TNF- a凋亡信号通路” Fas信号通路 7 - 9。 IAPs 是由一组有凋亡特性的结构相关的蛋白质构成 10，在预防肿瘤细胞凋 亡方面可能扮演一个重要的角色 , 并已成为近年来研究的热点。 这个家庭的新成员livin11-14。有证据表明 , livin 的过分表达能阻止 由多种刺激诱导的细胞凋亡 12 。有趣的 是, livin 基因被发现在肿瘤细胞中限制性表达 , 但是不存在或是很少数量存在于 正常成人体组织中 11-15 , 并且通过允许恶性细胞 ,以避免凋亡细胞死亡的方式导致肿瘤形成 , 。所以

6、抑制 livin 基因表达可能会呈现出一个有趣的治疗策略。 在目前的研究中 , 我们调查了 livin 的表达在胃 cancinomas 及其邻近组织。 livin 的表达和临床病理参数之间的关系进行了分析。此外 , 我们探索了在抑制 livin 基因表达的shRNA可行性和胃癌易感性的凋亡细胞由shRNA介导的livin沉默基因。2 患者和方法患者和肿瘤样本胃癌中四十个病例及接受胃切除手术的患者收集到的胃癌组织中 13 个病例 （患者年龄从 2977岁）。其中良性胃溃疡的 13个病例（慢性浅表胃炎 ）患者在接受 了胃内视镜检查 （患者年龄从 3377岁）。这些病例均来自南京医科大学第一附属

7、医院。胃癌患者被诊断为TNM级的14阶段（UICC，2002）。手术之后肿瘤标本就 立即被冻结在液态氮中，储存在-80C直到使用为止。这是在所有的病人知情同意 的情况下获得的。逆转录聚合酶链反应技术程序总共RNA（2毫克）提取冷冻组织反转录进行，最后的体积2微升是用100 pmolof oligo（dT）15 和200UM-MLV与逆转录酶（promega、美国）,根据制造商的说明。Aliquots对应的250微升cDNA被放大在PCR缓冲容器中，在最终的50微升 中含25pmol / ml处理剂和1U聚合酶。每一个放大了 35周期,一个周期的变性曲 线在30 s内到达94C。热处理在 30s

8、内59C（ livin and b-actin ）扩大到30s 内72C。没有RNA勺病例作为阴性对照物包含在 RT- PCR中。一系列常用的的 livin 和 B -actin 处理剂如下： livin a / B upstream，5 -TCCACAGTGTGCAGGAGACT-3 ;livin a / B downstream;5 -TCCACAGTGTGCAGGAGAC;TB-3-actin upstream,5 -ACGGCACAAAGACGATGGAC-3 B -actin downstream,5 ' -AGCGCAAGTACTCCGTGTG-产品 的尺寸分别为 livi

9、n a /B 是 312/258 bp ，B -actin 是 501bp。西方吸干技术分析病变同质性与冲力缓冲 50mMTris-HCI （pH , 250 mMNaCl,% NP40和 5mMEGTA 包含50mM氟化钠，60mMB -丙三醇-磷酸盐,0.5mM钒酸钠,0.1mM苯甲基磺醯化氟 10卩g/ml亮抑蛋白酶肽。用考马斯亮蓝微盘比色法测定蛋白质含量。蛋白质样品电泳10%变性SDS凝胶并转移到PVDF膜（Roche、美国）。 膜是培养特定的主要的抗体 , 与过氧化物酶继发性抗体反应 （ 细胞信号技术、美 国）, 最后通过增强化学荧光达到可视化 （ 细胞信号技术、 美国 ） 。 Al

10、exesis（ 美国） 和散塔克鲁兹生物技术 （ 美国 ） 购买的单克隆抗体 livin （1:250） and actin （1:400）24 细胞系和细胞培养我们选了一个人类胃腺癌细胞系在这一研究中。SGC-7901上海细胞研究所,中国上海 ,） 是一种附着中度分化胃腺的人类细胞株。 线是胃癌细胞上皮细胞 ,并成 长为附着细胞 RPMI 1640 （Hyclone Inc, USA） 含 10%FCS （Life Technologies, Inc.）, 每毫升 100 个单位的青霉素和毫升 100 微克的链霉素 （BioWhittaker） 。 在含有5%CO空气的条件下的37C湿润培养

11、器中保存SGC-7901细胞。溶解顺铂和 氟尿嘧啶（齐鲁制药厂、中国）在DMS（并且4C储存。ShRNA勺合成和PGPU / GFP /Neo/livin 质粒的制造通过siRNASequence-Selector软件设计并合成了 Livin的ShRN序列（上海生 物 技术 有限责任公司。 集 团公 司、 中 国 ） 。 序 列 如下 （ 表1）, 然后 被插 入 pGPU/GFP/Neo上海GenePharm股份有限公司。中国 ）Bbsl and BamH地址产生 pGPU/GFP/Neo/livin and pGPU/GFP/Neo/Control 质子。的建立稳定表达 pGPU/GFP/

12、Neo/livin and pGPU/GFP/Neo/Control 转染实 验,SGC-7901细胞被镀成6孔板（3 X 105孔密度）,96 孔板（1 X 104孔密度）和12孔 板X 105孔密度）转染之前培养24小时。按照制造商的说明这些细胞被调控子用 Li-pofectAMlNE 2000 转染4毫克/孔 空的 pGPU/GFP/Neo/vector, pGPU/GFP/Neo/livin 或pGPU/GFP/Neo/Control 质粒 （生命技术公司、大岛屿,NY）。转染48小时后，这些细胞被转移在1:15 （v/v）并用 Geneticin （G418） 1000克/毫升培养

13、4周。稳定转染的克隆体取出并保存在媒介容 器400 g/ml G418 用作另外的研究。27 依赖贴壁细胞细胞生长的测定亲本细胞和细胞稳定表达 pGPU/GFP/Neo/vector, pGPU/GFP/Neo/livin or pGPU/GFP/Neo/Control被种到6孔盘子中。每隔一天收集三孔的细胞。使用计数器 确定细胞数目 （Coulter Electronics, Miami, FL）.。种植一些天数后用平均 SD记录每孔细胞的数量。测定通过MTT对细胞毒性进行了测量。呈几何数增长的细胞被镀在密度为 10000 细胞/孔的 96 孔板上作用 24 小时。接下来这些细胞被以不同浓度

14、的药物治疗 48 小时。每孔加入100微升MTT溶液（1毫克/毫升）,并且这些细胞放在37E下培养 四小时。上层清液用异丙醇代替溶解有色甲品。用 micro-ELISA 测量到吸光率的 波长为 595nm（ClinBio-128 SLT, , 奥地利 ） 。治疗细胞的吸光率相当于计算控制细 胞的吸光率并用细胞死亡百分率显示出来。流式细胞术细胞被收集并加入冰冷的70聽醇于PBS缓冲液中储存在-4摄C暖直到使用。 悬浮后后，100 ml核糖核酸酶I （1 mg/ml） and 100 ml的聚酰亚胺（400卩g/ml） 37°C 下培养细胞并用流式细胞术（BD,美国）进行分析。统计分析数

15、据的展现要用至少三个不同实验土 SD的方法。实验结果用学生的t检验来 分析和当 P < 时被认为是具有统计学显著性。3 结果。 livin 在胃肠癌中的表达在目前的研究中 , 我们第一次验证了逆转录聚合酶链反应技术和西方吸干技 术的存在在 40胃癌中,13 癌组织和 13良性病变胃粘膜损伤。在癌组织和良性病 变胃粘膜损伤中,每个mRN亚型不可见水平被发现后，在肿瘤组织中，19/40%）显 示出 mRNAi livina a 和 livin B 蛋白质表达（Figs. 1 and 2）.。livin 表达与预后变量相关 ， 如组织学恶性度和淋巴结转移 ， 但包括年龄、性别、阶段和肿瘤细 胞

16、浸润程度 （表2）。稳定转染表达 pGPU/GFP/Neo/livin 与pGPU/GFP/Neo/Control 的特征我们建立了 SGC-7901 与任一 pGPU/GFP/Neo/livin 稳定转染， pGPU/GFP/Neo /Control 质粒，或空 pGPU/GFP/Neo/vector （ 图3）。用西方吸干技术和逆转录聚 合酶链反应技术选择每个转染的克隆并分析决定 livin mRNA， 和蛋白质表达。其他的都被选择作为扩展和另外的研究。（如图4及5）显示，livinmRN和蛋白质的水平在SGC-7901pGPU/GFP/Neo/livin2中转染降低了 90%以上。Liv

17、in表达抑 制在pGPU/GFP/Neo/livi n1转染和消极控制中没有被发现。所以 SGC-7901 pGPU/GFP/Neo/livin2 被用来做后续实验。稳定转染中抑制细胞生长SGC-7901的增长率pGPU / GFP /Neo/ livin2 均有显著的抑制转染。如图显 示，SGC-7901 Pgpu/GFP尼欧/ livin2 / GFP细胞数目有显著下降转染在 72小时到 96 小时后镀 （P < ， 而阴性对照和家长的细胞。稳定的转染容易受细胞凋亡因子刺激我们认为 SGC-7901 pGPU/GFP/Neo/livin2 转染和阴性对照细胞同细胞毒顺 铂的增长速率明

18、显抑制， 图表 6中显示 SGC-7901pGPU/GFP/Neo/livin2 转染细胞 数量培养后 72 小时和 96 小时与消极控制和亲本细胞相比明显降低阴性对照细胞和细胞毒药物（5 -氟尿嘧啶和顺铂）。pGPLMTT测定表明，SGC-7901/Neo/ livin2 / GFP更敏感转染顺铂和氟尿嘧啶比消极的控制和亲本细胞（Figs. 7A, 6B）。由顺铂和 5 氟尿嘧啶诱导凋亡细胞数增加至约 - 3 倍的 pGPU/GFP/Neo/livin2 转相 比，其控制细胞（P<图7C条。）。此外，经历了稳定转染无自发性凋亡更容易比 对照细胞（Pv图7C条）凋亡刺激。讨论在本研究中

19、, 我们表明 , 推荐的新成员 : 一个新的 IAP 家族成员，被认为是不 符合非癌胃组织的表达， 只有在胃癌患者 （）的比例计算， 也表明， 抑制 Livin 的表达或功能的原因自发性细胞凋亡和对 SGC- 7901 细胞生长的抑制，使细胞更 容易凋亡刺激。据认为，活着有两个亚型，A和B虽然这两个亚型在阻止肿瘤坏死因子诱导细胞凋亡参与-A和抗CD95的在体外，他们表现出一些不同的抗凋亡 的特性。活着b似乎是在阻断DNA损伤剂诱导细胞凋亡13超过活着有效。一些组织中Livin分布研究表明，最近都活着升高亚型A和B已发现在心脏， 胎盘，肺，脾，卵巢，而活着 balone 特别是在已检测到胎儿组织

20、和 dult 肾脏和 Livin 一单是在脑，骨骼肌和外周血淋巴细胞的检测 11-14 。此外，虽然 Livin 的表达是在一个癌细胞的细胞株和肿瘤组织中的一些品种检测 14-18 和反活着 抗体在胃癌和肺癌患者血清识别 19,20 ，没有数据有关亚型中 Livin 的表达在胃 癌肿瘤组织。我们的第一次研究表明，活着亚型 A和B几乎都在胃癌组织（）和 Livin 表达与一些已知的预后因素，如分级，淋巴结转移，相关的比例计算。从文献资 料表明，这两个活着亚型参与了阻止细胞凋亡，并可能给活着的过度表达与细胞 的强烈抵抗化疗诱导细胞凋亡。 胃癌一般具有高度抗癌症放化疗和中抗凋亡 21 。 这些结果表

21、明， Livin 的高表达可能对某些癌症患者和胃癌患者预后化疗的责任。与促进肿瘤细胞的凋亡抵抗可能提供一个合理干预策略在癌症治疗的基础上 发展新的特定因素干扰 22,23 。由于 Livin 的表达可能有助于肿瘤细胞和肿瘤的 特异性表达及其在细胞凋亡的抗性表型可以让活着的一个有趣的肿瘤治疗靶点的 具体干预措施的战略，我们选择了作为一个分子靶点的Livin基因。的shRNA技术 representiong 一个极其有力的工具，抑制内源性基因表达 24,25 作出抑制 Livin基因，并试图纠正胃癌细胞凋亡的不足。作者：沉默的shRNA功效的tageted 基因的表达是不同的，与半的生活和丰富的基

22、因产物与靶mRN作为24-27，以及无障碍的关系。在这项研究中，我们观察到硅 livin1 是经常更强烈的沉默比硅 livin2 Livin 基因。我们的研究结果还表明，沉默 Livin 基因的表达可能存在强烈的增加或几 个凋亡的代理人在场下的 SGC - 7901 细胞凋亡反应，抑制细胞的生长，这表明， 与美好生活的干扰导致了对凋亡刺激的敏感性。对 HeLa 细胞类似的结果报告了 Crnkovic - 梅坦斯 18 。总之，我们的结果表明， Livin 的表达和功能抑制自发性细胞凋亡和抑制细 胞生长的体外敏感性增强化疗药物的结果。由于在胃癌中的表达，但活着的优惠 在正常组织中，这些数据表明，

23、针对活着途径单独或与细胞毒性药物可能在胃癌 的治疗作用。尽管他们的治疗潜力，主要技术障碍仍有待克服，才能申请成为毒 品的 shRNA。在治疗方面，将不得不满足基因治疗的办法，如高效输送到目标细胞的免疫 反应或规避，一般的挑战。值得注意的是，在最近的研究表明，体内的shRNA可以直接应用到出生后小鼠脏器 highpressure 尾静脉注射，导致靶基因特异性抑制 28-30。这些数据表明，一个活跃的shRNA通过血液的直接应用是主要可行的。英文翻译：对照版Expression of livin in gastric cancer and induction of apoptosis in SGC

24、-7901 cells by shRNA-mediated silencing of livin geneBackground-Because of increased resistance to apoptosis in tumor cells, inhibition of specific antiapoptotic factors may provide a rational approach for the development of novel therapeutic , a novel inhibitor of apoptosis protein family, has been

25、 found to be expressed in various malignancies and is suggested to have poorly prognostic significance. However, no data are available concerning the significance of livin in gastric cancer. In this study, we detected the expression of livin in human gastric carcinoma and investigated the apoptotic

26、susceptibility of SGC-7901 cell by shRNAmediated silencing of the livin gene.Methods-The mRNA and protein expression of livin were analyzed by RT-PCR and western blot relationship between livin expression and clinical pathologic parameters was investigated. The small interfering RNA eukaryotic expre

27、ssion vector specific to livin was constructed by generecombination, and the nucleic acid was sequenced. Then it was transfected into SGC-7901 cells by Lipofectamin 2000. RT-PCR and Western blot assay were used to validate gene-silencing efficiency of livin in SGC-7901cells. Stable clones were obtai

28、ned by G418 screening. The cell apoptosis was assessed by flow cytometry (FCM). Cell growth state and 50 % inhibition concentration (IC50) of 5-FU and cisplatin was determined by MTT method.Results-The expression of livin mRNA and protein were detected in 19 of 40 gastric carcinoma cases %) and SGC-

29、7901cells. No expression of livin was detected in tumor adjacent tissues and benign gastric lesion. The positive correlation was found between livin expression and poor differentiation of tumors as well as lymph node metastases (P < . Four small interfering RNA eukaryotic expression vector specif

30、ic to livin were constructed by gene recombination. And one of them can efficiently decrease the expression of livin, the inhibition of the gene was not less than 70% (P < . The recombinated plasmids were extracted and transfected gastric cancer cells. The stable clones were obtained by G418 scre

31、ening, and were amplified and cultured. When livin gene was silenced, the reproductive activity of the gastric cancer cells was significantlylower than the control groups(P < . The study also showedthat IC50 of 5-Fu and cisplatin on gastric cancer cells treated by shRNA was decreased and the cell

32、s were more susceptible to proapoptotic stimuli (5-Fu and cisplatin) (P < .Conclusions-C Livin is overexpressed in gastric carcinoma with a relationship to tumor differentiation and lymph node metastases, which is suggested to be one of the molecular prognostic factors for some cases of gastric c

33、ancer. ShRNA can inhibit livin expression in SGC-7901 cells and induce cell may serve as a new target for apoptosis-inducing therapy of gastric cancer.1. IntroductionGastric cancer is one of the most common malignancies in the world.Most patients with this disease are diagnosed in advanced stages, a

34、nd lose the chance of surgical eradication. Despite muchprogress in chemotherapy, the overall survival of the patients with gastric cancer in advanced stage is still poor. Resistance of cancer cells to chemoagents may contribute to failure of the treatment. Among the reasons of drug resistance, inhi

35、bited process of cell apoptosis may play an important role.Cancer cells are often characterized by increased resistance to apoptosis 1, which mediates their increased resistance to various stimuli of cell apoptosis, such as DNA damage, hypoxia, nutrient-deprivation 2,3. Moreover, apoptosis resistanc

36、e is considered to be a major cause of therapeutic failure for tumors in clinical practice, since many chemo- and/or radiotherapeutic agents function through the induction of apoptotic tumor death 4.Inhibitor of apoptosis protein (IAPs) is a novel family of intracellular proteins which suppress apop

37、tosis induced by a variety of stimuli 5,6, including viral infection, chemotherapeutic drugs, staurosporin, growth factor withdrawal, and by components of the tumornecrosis factor-a (TNF-a)/Fas apoptotic signaling pathways 7C9. TheIAPs consists of a group of structurally related proteins with antiap

38、optotic properties 10, and may play a substantial role in preventing tumor cell from apoptosis, and has becomethe focus of research in recent years. A novel memberof this family is ML-IAP/livin/KIAP/BIRC7 (in the following termed livin) which has two isoforms, livin a and livin b 11 " C14. It h

39、as been shown that over -expression of the livin can block apoptosis induced by a variety of proapoptotic stimuli 12. Interestingly, livin gene has been found to be restrictively expressed in tumor cells,but not, or to lesser amounts in most normal adult tissues 11 C15, and may contribute to tumorig

40、enesis by allowing malignant cell to avoid apoptotic cell death. So inhibition of livin expression may represent an interesting therapeutic strategy.In the present study, we investigated the expression of livin in gastric cancinomas and their adjacent tissues. The relationship between livin expressi

41、on and clinical pathologic parameters was analyzed. Furthermore, we explored the feasibility of shRNAin inhibiting livin gene expression and the apoptotic susceptibility of gastric cancer cell by shRNA-mediated silencing of the livin gene.2. Patients and methods. Patients and tumor samplesForty samp

42、les of gastric carcinoma and 13 samples of paracancerous tissues were collected from the patients who received gastrectomy (age of patients ranging from 29-77 years). Thirteen samples of benign gastric lesion (chronic superficial gastritis) were gained from the patients undergoing gastric endoscopic

43、 examination (age of patients ranging from 33-77 years). These samples were collected from patients admitted to the First Affiliated Hospital of Nanjing Medical University. The patients with gastric cancer were diagnosed as being in stage I to IV based on TNM classification (UICC, 2002). Tumor speci

44、mens were immediately frozen in liquid nitrogen after surgery and stored at -80°C unt il use. Informedconsent was obtained from all patients. RT-PCR procedureTotal RNA(2 mg) extracted from frozen tissues was reverse transcribed in a final volume of 25 ml with 100 pmol of oligo(dT)15 and 200U M-

45、MLV reverse transcriptase (promega, USA), according to the manufacturer'sguidelines.Aliquots corresponding to ml cDNAwere then amplified in PCRbuffer containing 25pmol/ml each primer and 1 U Taq polymerase in a final volume of 50 ml. Each amplification was performed for 35 cycles, one cycle prof

46、ile consisted of denaturationat 94 8C for 30 s, annealing at 59 8C (livin and b-actin) for 30 s and extension at 72 8C for 30 s. A sample without RNA was in eluded in each RTCPCR as a n egative con trol.Seque nces of livi n and&act in primers used are as follows:livi na/bupstream,50-TCCACAGTGTGC

47、AGGAG3A0C;iTv-ina/B downstream,50-ACGGCACAAAGACGATGGAC-30;b-acti nu pstream,50-AGCGCAAGTACTCCGTG0fQB-actin downstream, size of the amplified products were312/258 bp for livina/b and 501 bp for b-actin respectively. Western Blot AnalysisTissues were homogenized with lysis buffer 50 mM Tris-HCl(pH , 2

48、50 mM NaCl, % NP40, and 5 mM EGTA containing 50 mM sodium fluoride, 60 mM b-glycerol-phosphate, 0.5 mMsodium vanadate, 0.1 mMphenylmethylsulfonyl fluoride, 10 mg/ml aprotinin, and 10 mg/ml leupeptin. The total protein concentrationwas determined using Coomassie Brilliant Blue. Protein samples were e

49、lectrophoresed in a 10% denaturing SDS gel and transferred to PVDF membrane(Roche, USA). The membraneswere incubated with specific primary antibodies, reacted with a peroxidase-conjugated secondary antibody (Cell signaling technology,USA), and finally visualized by enhanced chemiluminescence (Cell s

50、ignaling technology, USA). Monoclonal antibodies recognizing livin (1:250) and actin (1:400) were purchased from Alexesis Inc. (USA) and Santa Cruz Biotechnology (USA). Cell lines and cell cultureWe selected a human gastric adenocarcinoma cell lines for thisstudy.SGC-7901 (Shanghai Institute of Cell

51、 Research, Shanghai,China) is an adherent, moderately differentiated, human gastric adenocarcinoma cell line. The cell lines are gastric cancer epithelial cells and grow as adherent cells in RPMI 1640 (Hyclone Inc, USA)containing 10% FCS (Life Technologies, Inc.), 100 units/ml penicillin,and 100 mg/

52、ml streptomycin (BioWhittaker). SGC-7901 cells were maintainedat37 8Cina humidified incubatorwithanatmosphere of 5% CO2. Cisplatin and 5-fluorouracil (Qilu pharmaceutical factory,China) were solublized in DMSOand stored at 4 8C. . ShRNA synthesis and construction of PGPU/GFP/Neo/livin plasmidsShRNA

53、sequences of livin were designed by software of siRNA Sequence-Selector and synthesized (Shanghai Biotech, Ltd.Corp., China). The sequences as following (Table 1)then were inserted into BbsI and BamH sites of the pGPU/GFP/Neo(Shanghai GenePharma Co. Ltd China) to generate pGPU/GFP/Neo/livin and pGPU

54、/GFP/Neo/Control plasmids,respectively. . Establishment of SGC-7901 stable transfectants expressing pGPU/GFP/Neo/livin and pGPU/GFP/Neo/ControlFor transfection experiments, SGC-7901 cells were plated into 6-well4plates (3? a 105cells/well),96- well plates (1 x 10 cells/well) and 12-well5plates x 10

55、cells/well) for 24 h before transfectionThe cells were transfected with 4 mg/well of empty pGPU/GFP/Neo/vector, pGPU/GFP/Neo/livin or pGPU/GFP/Neo/Control plasmid using Li-pofectAMINE 2000 (Life Technologies, Inc., Grand Island,NY) according to the manufacturer? - s instructions. Forty-eight hours a

56、fter transfection, thecells were passaged at 1:15 (v/v) and cultured in mediumsupplemented withGeneticin (G418) at 1000 g/ml for 4 weeks. Stably transfected clones were picked and maintained in medium containing 400 g/ml G418 for additional studies. Assay of anchorage-dependent cell growthParent cel

57、ls and cells stably expressing empty pGPU/GFP/Neo vector, pGPU/GFP/Neo/livin or pGPU/GFP/Neo/Control were seeded into 6-well plates. Cells from triplicate wells were collected every other day. Cell numbers were determined using a Coulter counter (Coulter Electronics, Miami, FL). The number of cells

58、per well is reported as the average SDat the indicated number of days after plating. MTT assayCytotoxicity was measured by MTT assay. Cells growing exponentially were plated onto 96-well plates at a density of 10000 cells/well for 24 h. The cells were then treated with different concentrations of dr

59、ugs for 48 h. One hundred microliters of MTT stock solution (1 mg/ml) were addedto each well, and the cells were further in cubated at 37°C for 4 h. T hesupernatant was replaced with isopropyl alcohol to dissolve formazan production.The absorbance at wavelength 595 nm was measured with amicro-ELISA reade