地西他濱聯(lián)合順鉑逆轉人胃癌SOX2基因甲基化狀態(tài)的實驗研究
發(fā)布時間：2019-03-02 20:22
【摘要】：研究目的:近年來的研究發(fā)現(xiàn),SOX2與胃癌的發(fā)生發(fā)展及其惡性生物學行為顯著相關。本課題擬在前期研究工作的基礎上,探討地西他濱聯(lián)合順鉑對人胃癌細胞以及裸鼠移植瘤模型中SOX2基因甲基化狀態(tài)的影響,明確SOX2基因的甲基化狀態(tài)、mRNA和蛋白表達水平與人胃癌細胞以及移植瘤生長增殖的關系。方法:體外培養(yǎng)人胃癌細胞株BGC-823至對數(shù)生長期,地西他濱、順鉑單獨或聯(lián)合用藥,WST-1方法分析藥物處理對細胞生長增殖的影響,流式細胞術檢測細胞凋亡情況,Transwell實驗檢測細胞的體外遷移能力。同時,MSP技術檢測細胞中SOX2基因啟動子甲基化情況,RT-PCR、Western blot分別檢測SOX2基因的mRNA和蛋白表達水平。然后,建立人胃癌裸鼠移植瘤模型,地西他濱、順鉑單獨或聯(lián)合給藥,計算各組移植瘤的體積并繪制生長曲線。給藥完成后,取移植瘤組織,免疫組化法檢測各組移植瘤組織Ki-67分子的表達水平,MSP技術檢測SOX2基因的甲基化情況,RT-PCR、Western blot分別檢測SOX2基因的mRNA和蛋白表達水平。結果:在體外細胞實驗中,地西他濱(5μM)、順鉑(1μM、10μM、100μM)以及兩者的聯(lián)合應用均能以時間和(或)劑量依賴性的方式抑制人胃癌細胞BGC-823的生長增殖和遷移能力,均能一定程度地引起SOX2基因啟動子區(qū)域DNA的去甲基化,增加SOX2基因的m RNA和蛋白表達量,而地西他濱聯(lián)合順鉑則能協(xié)同發(fā)揮上述作用。在人胃癌裸鼠移植瘤模型中,地西他濱(5 mg/kg)、順鉑(6 mg/kg)對移植瘤的生長均有一定的抑制作用,而聯(lián)合用藥則能明顯抑制移植瘤的生長和增殖能力。類似的,地西他濱和順鉑也均一定程度地增加了移植瘤組織中SOX2基因的去甲基化狀態(tài),上調了SOX2基因的mRNA和蛋白表達水平,而地西他濱聯(lián)合順鉑則能協(xié)同發(fā)揮上述作用。結論:在體外細胞水平和人胃癌裸鼠移植瘤模型中,地西他濱聯(lián)合順鉑能協(xié)同逆轉SOX2基因的甲基化狀態(tài),重新恢復其mRNA和蛋白的表達,從而在抑制細胞生長增殖和遷移、誘導細胞凋亡、抑制移植瘤的生長和惡性增殖能力等過程中協(xié)同發(fā)揮抗腫瘤效應。
[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.
【學位授予單位】：福建醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R735.2
[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.
【學位授予單位】：福建醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R735.2
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