【作者】 朱晓东；

【导师】 林庚金；

【作者基本信息】 复旦大学 ， 内科学， 2003， 博士

【摘要】 Survivin是近年来发现的凋亡抑制蛋白家族（IAP family）的新成员，能通过与Caspase3、7等的结合抑制Caspase活性而抑制凋亡。研究表明survivin在凋亡的发生中起极重要的调控作用，同时survivin表达又与肿瘤的发生，一些患者的预后不良，生存率降低有关，还有研究认为survivin可能是良好的抗肿瘤治疗靶点。紫杉醇是从红豆杉中提取的抗肿瘤药物，目前的研究表明该药对胃癌等消化系统肿瘤有很好的应用前景，其主要抗肿瘤机理之一是诱导肿瘤细胞凋亡，故紫杉醇极可能会影响到survivin的表达。但紫杉醇能否通过抑制survivin表达来诱导凋亡目前尚无报道，与凋亡密切相关的JNK、p38蛋白激酶通路对survivin表达有无调控亦不清楚，并缺乏以survivin为靶点的体内基因治疗研究。我们研究survivin在紫杉醇抗肿瘤机制中的作用将有助于进一步明确紫杉醇的抗肿瘤机理；采用脂质体介导的体内转染法，研究survivin反义寡核苷酸及联合紫杉醇对实验性大鼠胃癌形成的影响，探讨以survivin为靶点进行体内抗肿瘤基因治疗的效果和可行性，可以为基因治疗早日进入临床应用提供理论和实验基础。第一部分 survivin在人胃癌中的表达与临床病理及p53、c-erbB2表达的关系目的 研究人胃癌组织中survivin蛋白表达与临床病理及与p53和c-erbB2表达的关系。方法 采用免疫组化的方法检测56例胃癌患者肿瘤组织及20例慢性胃炎患者胃粘膜中survivin和p53、c-erbB2的表达。结果 20例慢性胃炎患者胃粘膜中survivin的表达阳性1例，阳性率为5％；56例胃癌组织中survivin阳性27例，阳性率48.2%；56例胃癌患者c-erbB2阳性率35.7%（20/56），p53阳性率39.3%（22/56）。survivin表达与患者年龄、肿瘤浸润深度、淋巴结转移、临床分级等无明显相关性，而与肿瘤Lauren组织分型相关。survivin 阳性率在肠型胃癌（20/32，62.5%）中显著高于弥漫型（7/24，29.2% P<0.05）。survivin 在c-erbB2阳性和阴性患者中阳性率分别为80%（16/20）和30.6％（11/36），两者间有显著差异（P<0.01）；survivin 在 p53阳性和阴性患者中阳性率分别为68.2%（15/22）和35.3%（12/34），两者比较亦有显著差异（P<0.05）。结论 胃癌患者survivin的异常表达在胃癌的发生发展中起了重要作用，survivin表达与胃癌中p53和c-erbB2异常表达相关。第二部分 紫杉醇对胃癌细胞survivin表达的影响及其调控机理目的 研究紫杉醇诱导胃癌细胞凋亡过程中对survivin表达的影响及其中的调控机理。 方法 采用Western blot的方法检测五株胃癌细胞中survivin表达，用<WP=5>MTT方法检测紫杉醇对各株细胞的IC50，分析survivin表达与IC50的关系；检测紫杉醇干预后72小时内survivin、JNK和p38激酶表达的变化，并检测分别预先采用curcumin和SB203580抑制JNK和p38激酶通路后survivin表达的改变；用Western blot检测89kDa-PARP的方法和TUNEL法检测抑制剂使用前后凋亡的变化。 结果 紫杉醇对五株胃癌细胞的IC50与细胞的survivin表达正相关；在60ng/ml紫杉醇作用下，五株胃癌细胞中的survivin在24小时内均有不同程度的升高；600ng/ml紫杉醇能导致survivin表达在24小时内有较明显的升高（增幅近70％），而36至72小时内明显下降，而紫杉醇干预后JNK明显升高，一直持续到48小时，p38激酶的磷酸化在1、3小时时明显升高；预先以curcumin抑制JNK激酶，survivin的表达升高更为显著（增幅130％），89kDa-PARP高峰后移， TUNEL示凋亡率下降；而预先以SB203580抑制p38激酶则survivin表达不再明显升高，89kDa-PARP提前出现，TUNEL示凋亡率有所增高。 结论 紫杉醇对胃癌细胞株的IC50与细胞中survivin表达呈正相关；紫杉醇能促进JNK表达和p38磷酸化，JNK介导了凋亡的发生，并能抑制survivin表达，而p38介导凋亡抑制并促进survivin表达；紫杉醇诱导的凋亡与其激活JNK最终导致survivin下降有一定的关系，而p38介导的凋亡抑制作用与其使survivin表达升高有关。第三部分 survivin反义寡核苷酸的抗肿瘤作用和与紫杉醇的协同作用目的 研究survivin反义寡核苷酸及联合紫杉醇对胃癌细胞生长增殖的影响。 方法 以脂质体包裹survivin反义或正义寡核苷酸瞬时转染胃癌BGC-823细胞，以MTT法检测寡核苷酸转染后及联合紫杉醇时对细胞生长增殖的抑制作用。 结果 survivin正、反义寡核苷酸均能顺利转入BGC-823细胞，在胞核和胞浆均见到绿色荧光；survivin反义寡核苷酸能明显抑制survivin蛋白的表达，并抑制BGC-823的生长，其IC50约350nM左右，survivin正义寡核苷酸对BGC-823的生长影响不大； 紫杉醇与survivin反义寡核苷酸联合能显著降低其IC50值，起到协同抗肿瘤作用。 结论 survivin反义寡核苷酸能抑制BGC-823细胞生长，和紫杉醇有协同抗肿瘤作用。第四部分 survivin在实验性大鼠胃癌中的表达及survivin反义寡核苷酸联合化疗药物对胃癌的干预作用目的 研究在胃癌发生过程中survivin表达的变化及survivin反义寡核苷酸联合化疗药物对胃癌形成的干预作用。 方法 121只六周龄的雄性Wistar大鼠随机分为正常组和模型组，正常对照组16只不作任何处理；模型组105只，给予含更多还原

【Abstract】 Survivin , a new member of IAP family, can inhibit apoptosis by combining with Caspase3 and Caspase7. Sufficient data show that survivin plays an important role in apoptosis pathway and that its’ expression is highly related with cancer and associated with an unfavorable prognosis. Some study indicated that survivin may be a promising target of anticancer therapy. Taxol is a compound extracted from Yew and clinical data has proved that taxol is a promising drug for the chemotherapy of gastrointestinal cancer. Inducing apoptosis is an important antitumor mechanism of Taxol, hence Taxol may have an influence on the expression of survivin. Studying the role of survivin in Taxol’s effects will contribute to clarify the antitumor mechanism of Taxol. With the transfection mediated by Lipofect Amine, we studied effects of survivin antisense and it’s combination with Taxol on the development of experimental gastric cancer in order to evaluate the effect of survivin targeted gene therapy in vivo.Part 1: The expression of survivin in gastric carcinoma, and it’s relationship with the expression of p53 and c-erbB2 Objective: To study the expression of survivin in gastric carcinomas and its relationship with the expression of p53, c-erbB2.Method: Using immunohistochemical staining method, we examined the expression of survivin, p53 and c-erbB2 genes in 20 cases of chronic gastritis and 56 cases of gastric carcinomas.Results: Survivin expressed in 27 of 56(48.2%) cases of gastric carcinoma tissues and 1 of 20(5%) cases of chronic gastritis. Over expression of survivin had no relation with age, tumor depth, tumor size, and disease stage, but was significantly related to histological type. The expression of survivin significantly segregated with intestinal type cases as compared with diffuse type cases. Survivin positive cases were significantly associated with p53 expression(15/22,68.2% versus 12/34,35.3%,P<0.05) and c-erbB2 expression(16/20,80% versus 11/36,30.6%,P<0.01).Conclusions: These data indicated that survivin play a important role in the onset of gastric cacinoma and suggested potential correlation between survivin and c-erbB2, and between survivin and p53 as well.<WP=8>Part2: Regulation on survivin expression in gastric cancer cells by Taxol and it’s mechanismObjective: To study the regulation of Taxol on the expression of survivin in gastric cancer cells and it’s mechanism. Method: Survivin expression of five gastric cancer cell lines were examined by western blot, and the IC50 of Taxol to each cell line was detected by MTT. We also examined survivin expression of each cell line after treated with 60ng/ml Taxol in 24 hours, and examined survivin expression in BGC-823 cell line after treat with 600ng/ml Taxol in 72 hours. The expression of JNK kinase, phosphorylation of JNK kinase and phosphorylation of p38 kinase were detected simultaneously. In addition, SB203580 and curcumin were used to inhibit p38 and JNK pathway respectively before treatment with Taxol for 72 hours, then survivin expression were examined by western blot, and cell apoptosis were detected by TUNEL and 89kDa-PARP. Result: The IC50 of Taxol to these five gastric cell lines were correlated with survivin expression. Survivin expression of these five cell lines increased to some extend respectively during the 24 hours after 60ng/ml Taxol treatment. When BGC-823 cells were treated with 600ng/ml Taxol, the expression of survivin increased 7 fold at 24 hour and then decreased. JNK kinase expression increased after Taxol treatment and remain at a high level for about 48 hours, and phosphorylation of p38 kinase increased from 1 to 3 hour after Taxol treatment. Pretreated BGC-823 with curcumin 10uM for 1 hour before Taxol treatment, survivin expression increased 1.3 time in 24 hour, the peak of 89kDa-PARP delayed, and apoptosis index examined by TUNEL decreased. When pretreated with SB203580 10uM for 1 hour, no increase of survivin expression was detected during 24 hour, the presence of 89kDa-PARP was brought forward,更多还原