地西他滨联合顺铂逆转人胃癌SOX2基因甲基化状态的实验研究

发布时间：2019-03-02 20:22

【摘要】：研究目的:近年来的研究发现,SOX2与胃癌的发生发展及其恶性生物学行为显著相关。本课题拟在前期研究工作的基础上,探讨地西他滨联合顺铂对人胃癌细胞以及裸鼠移植瘤模型中SOX2基因甲基化状态的影响,明确SOX2基因的甲基化状态、mRNA和蛋白表达水平与人胃癌细胞以及移植瘤生长增殖的关系。方法:体外培养人胃癌细胞株BGC-823至对数生长期,地西他滨、顺铂单独或联合用药,WST-1方法分析药物处理对细胞生长增殖的影响,流式细胞术检测细胞凋亡情况,Transwell实验检测细胞的体外迁移能力。同时,MSP技术检测细胞中SOX2基因启动子甲基化情况,RT-PCR、Western blot分别检测SOX2基因的mRNA和蛋白表达水平。然后,建立人胃癌裸鼠移植瘤模型,地西他滨、顺铂单独或联合给药,计算各组移植瘤的体积并绘制生长曲线。给药完成后,取移植瘤组织,免疫组化法检测各组移植瘤组织Ki-67分子的表达水平,MSP技术检测SOX2基因的甲基化情况,RT-PCR、Western blot分别检测SOX2基因的mRNA和蛋白表达水平。结果:在体外细胞实验中,地西他滨(5μM)、顺铂(1μM、10μM、100μM)以及两者的联合应用均能以时间和(或)剂量依赖性的方式抑制人胃癌细胞BGC-823的生长增殖和迁移能力,均能一定程度地引起SOX2基因启动子区域DNA的去甲基化,增加SOX2基因的m RNA和蛋白表达量,而地西他滨联合顺铂则能协同发挥上述作用。在人胃癌裸鼠移植瘤模型中,地西他滨(5 mg/kg)、顺铂(6 mg/kg)对移植瘤的生长均有一定的抑制作用,而联合用药则能明显抑制移植瘤的生长和增殖能力。类似的,地西他滨和顺铂也均一定程度地增加了移植瘤组织中SOX2基因的去甲基化状态,上调了SOX2基因的mRNA和蛋白表达水平,而地西他滨联合顺铂则能协同发挥上述作用。结论:在体外细胞水平和人胃癌裸鼠移植瘤模型中,地西他滨联合顺铂能协同逆转SOX2基因的甲基化状态,重新恢复其mRNA和蛋白的表达,从而在抑制细胞生长增殖和迁移、诱导细胞凋亡、抑制移植瘤的生长和恶性增殖能力等过程中协同发挥抗肿瘤效应。
[Abstract]:Objective: in recent years, it has been found that SOX2 is associated with the occurrence and development of gastric cancer and its malignant biological behavior. The purpose of this study was to investigate the effects of desetabine combined with cisplatin on the methylation status of SOX2 gene in human gastric cancer cells and xenograft tumor model in nude mice, and to clarify the methylation status of SOX2 gene. The relationship between the expression of mRNA and protein and the growth and proliferation of human gastric cancer cells and transplanted tumor. Methods: human gastric cancer cell lines BGC-823 were cultured in vitro from logarithmic phase to logarithmic growth period. The effects of drug treatment on cell growth and proliferation were analyzed by WST-1, and the apoptosis was detected by flow cytometry (FCM), and the effects of drug treatment on cell growth and proliferation were analyzed by using descitabine and cisplatin alone. The ability of cell migration in vitro was detected by Transwell assay. At the same time, the methylation of the promoter of SOX2 gene was detected by MSP, and the expression of mRNA and protein of SOX2 gene was detected by RT-PCR,Western blot. Then, the xenograft tumor model of human gastric cancer in nude mice was established. Descitabine and cisplatin were administered alone or in combination. The volume of transplanted tumor in each group was calculated and the growth curve was drawn. After administration, the expression of Ki-67 molecule was detected by immunohistochemistry, the methylation of SOX2 gene was detected by MSP technique, and the expression of mRNA and protein of SOX2 gene was detected by RT-PCR,Western blot. Results: desetabine (5 渭 M), cisplatin (1 渭 M, 10 渭 M, 100 渭 M) and the combination of desetabine (5 渭 M) and cisplatin (1 渭 M, 10 渭 M, 100 渭 M) could inhibit the proliferation and migration of human gastric cancer cell line BGC-823 in a time-and / or dose-dependent manner in vitro. Both of them could induce the demethylation of DNA in the promoter region of SOX2 gene to a certain extent, increase the expression of m-RNA and protein of SOX2 gene, and descitabine combined with cisplatin could play a synergistic role. In the xenograft tumor model of human gastric cancer in nude mice, desetabine (5 mg/kg) and cisplatin (6 mg/kg) could inhibit the growth of transplanted tumor to a certain extent, while the combination of desetabine and cisplatin could inhibit the growth and proliferation of transplanted tumor. Similarly, desetabine and cisplatin increased the demethylation of SOX2 gene and up-regulated the expression of mRNA and protein of SOX2 gene in transplanted tumor tissues to a certain extent, while descitabine combined with cisplatin could play a synergistic role in these effects. Conclusion: in vitro cell level and transplanted tumor model of human gastric cancer in nude mice, descitabine combined with cisplatin can co-reverse the methylation status of SOX2 gene and restore the expression of mRNA and protein, thus inhibiting cell growth, proliferation and migration. In the process of inducing apoptosis, inhibiting the growth of transplanted tumor and the ability of malignant proliferation, the anti-tumor effect is synergetic.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.2

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