洛铂联合紫杉醇对人胃癌SGC-7901细胞作用机制及细胞周期相关蛋白在卵巢癌组织中表达的研究

发布时间：2018-06-26 00:58

本文选题：洛铂 + 紫杉醇　；参考：《大连大学》2016年硕士论文

【摘要】：目的:探讨洛铂联合紫杉醇对人胃癌SGC-7901细胞的作用机制,并研究卵巢癌组织中AKAP95、cyclinE1、cyclinD1、Cx43蛋白的表达及与临床病理学指标的关系。方法:1、MTT实验分别检测洛铂、紫杉醇及两者联合作用于胃癌SGC-7901细胞系的抗肿瘤效应。2、运用流式细胞术检测洛铂、紫杉醇及两者联合应用对胃癌SGC-7901细胞的细胞周期分布影响。3、运用western blot方法检测洛铂、紫杉醇作用SGC-7901细胞后,cyclinD1/E1/B1及CDK2/4、Rb、pRb蛋白表达的变化。4、采用免疫组织化学方法检测54例卵巢癌组织中蛋白的表达。5、利用分子克隆技术构建cyclinE2过表达及沉默质粒。6、利用western blot和免疫荧光实验方法检测cyclinE2和LMW-cyclinE2的定位,并检测两种抗体对CCNE2编码的蛋白的特异性。7、利用Co-IP、银染、MS/MS方法鉴定cyclinE2(A-9)抗体对应蛋白是否为CCNE2基因编码的蛋白。结果:1、洛铂联合紫杉醇对胃癌SGC-7901具有协同作用联合指数CI1。2、联合使用洛铂和紫杉醇能增加SGC-7901细胞中cylinE1和CDK2的表达(P0.05),减少cylinB1、Rb、pRb的表达(P0.05),cyclinD1和CDK4表达变化无统计学意义(P0.05);联合使用可以导致细胞S期和M期阻滞。3、AKAP95、cyclinD1、cyclinE1蛋白在卵巢癌组织中表达的阳性率分别为72.22%、66.67%和79.63%,高于癌旁组织的33.33%、25.00%和8.30%;Cx43蛋白在卵巢癌组织的阳性率为40.74%,低于癌旁组织的75.00%,其差异均有统计学意义(P0.05)。4、在卵巢癌组织中,cyclinD1蛋白的表达与组织类型有关(P0.05),与分化程度无关(P0.05),AKAP95、cyclinD1、cyclinE1蛋白的表达与分化程度、组织类型均无关(P0.05)。5、AKAP95、cyclinE1和Cx43蛋白三者之间的表达均有关联性(P0.05),cyclinD1、cyclinE1和Cx43蛋白三者之间互有关联性(P0.05),而AKAP95与cyclinD1蛋白之间未见关联性(P0.05)。6、cyclinE2主要定位于细胞浆,核内少量表达,而LMW-cyclinE2主要定位于细胞核。7、cyclinE2对CCNE2编码的蛋白没有特异性,而LMW-cyclinE2对CCNE2编码的蛋白有特异性。8、cyclinE2(A-9)抗体对应的蛋白没有检测到CCNE2基因编码的氨基酸序列。结论:1、洛铂联合紫杉醇对胃癌SGC7901的细胞周期阻滞具有协同作用。2、AKAP95、cyclinD1和cyclinE1蛋白可能具有促进卵巢癌发生发生发展的作用,Cx43具有抑制卵巢癌发生发展的作用。3、cyclinE2(A-9)抗体识别未知蛋白,此蛋白可能不是LMW-cyclinE2,cyclinE2(40890)抗体识别的是cyclinE2蛋白。
[Abstract]:Objective: To investigate the mechanism of the action of luoplatin combined paclitaxel on human gastric cancer SGC-7901 cells and to study the expression of AKAP95, cyclinE1, cyclinD1, Cx43 protein in ovarian cancer and its relationship with the clinicopathological indexes. Methods: 1, MTT tests were conducted to detect the antitumor effect of luoplatin, paclitaxel and both of them on the anti-tumor effect.2 of gastric cancer SGC-7901 cell lines, respectively. Flow cytometry was used to detect the cell cycle distribution of SGC-7901 cells in gastric cancer by using luoplatinum, paclitaxel and both.3. The Western blot method was used to detect the changes of cyclinD1/E1/B1 and CDK2/4, Rb, and pRb protein expression in SGC-7901 cells of paclitaxel,.4, and 54 cases of ovarian cancer were detected by immunohistochemistry. The expression of medium protein.5, cyclinE2 overexpression and silencing plasmid.6 were constructed by molecular cloning technology. The localization of cyclinE2 and LMW-cyclinE2 was detected by Western blot and immunofluorescence assay, and the specificity of two antibodies against CCNE2 encoded proteins was detected. Co-IP, silver staining and MS/MS methods were used to identify the corresponding protein of the antibody. The protein encoded by CCNE2 gene. Results: 1, the combination of luoplatin and paclitaxel has synergistic combined index CI1.2. The combined use of luoplatin and paclitaxel can increase the expression of cylinE1 and CDK2 in SGC-7901 cells (P0.05), and reduce the expression of cylinB1, Rb, pRb (P0.05). The positive rates of.3, AKAP95, cyclinD1, cyclinE1 protein expression in ovarian cancer tissues were 72.22%, 66.67% and 79.63% respectively, higher than 33.33%, 25% and 8.30% in para cancerous tissues, and 40.74% for Cx43 protein in ovarian cancer tissue, and the difference was statistically significant (P0) (P0). The difference was statistically significant (P0) (P0). (P0), the positive rate of Cx43 protein in ovarian cancer tissues was 40.74%, lower than that of the para cancerous tissue (P0) (P0). .05).4, in ovarian cancer, the expression of cyclinD1 protein is related to the type of tissue (P0.05), not related to the degree of differentiation (P0.05), the expression of AKAP95, cyclinD1, cyclinE1 protein and the degree of differentiation (P0.05).5, AKAP95, cyclinE1, and egg white three There is a mutual association between the three (P0.05), and there is no association between AKAP95 and cyclinD1 protein (P0.05).6, cyclinE2 is mainly located in the cytoplasm and a small amount of expression in the nucleus, while LMW-cyclinE2 is mainly located in the nuclear.7, cyclinE2 is not specific to the protein encoded by CCNE2, but LMW-cyclinE2 on the proteins encoded by CCNE2. The antibody corresponding protein did not detect the amino acid sequence encoded by the CCNE2 gene. Conclusion: 1, luoplatin combined with paclitaxel has a synergistic effect on the cell cycle block of gastric cancer SGC7901,.2, AKAP95, cyclinD1 and cyclinE1 proteins may play a role in promoting the development of ovarian cancer. Cx43 has the effect of inhibiting the development of ovarian cancer,.3, cyc LinE2 (A-9) antibody recognizes unknown protein. This protein may not be LMW-cyclinE2, and cyclinE2 (40890) antibody recognizes cyclinE2 protein.
【学位授予单位】：大连大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.2

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