【摘要】：研究目的:近年來(lái)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(xué)行為顯著相關(guān)。本課題擬在前期研究工作的基礎(chǔ)上,探討地西他濱聯(lián)合順鉑對(duì)人胃癌細(xì)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(dá)水平與人胃癌細(xì)胞以及移植瘤生長(zhǎng)增殖的關(guān)系。方法:體外培養(yǎng)人胃癌細(xì)胞株BGC-823至對(duì)數(shù)生長(zhǎng)期,地西他濱、順鉑單獨(dú)或聯(lián)合用藥,WST-1方法分析藥物處理對(duì)細(xì)胞生長(zhǎng)增殖的影響,流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡情況,Transwell實(shí)驗(yàn)檢測(cè)細(xì)胞的體外遷移能力。同時(shí),MSP技術(shù)檢測(cè)細(xì)胞中SOX2基因啟動(dòng)子甲基化情況,RT-PCR、Western blot分別檢測(cè)SOX2基因的mRNA和蛋白表達(dá)水平。然后,建立人胃癌裸鼠移植瘤模型,地西他濱、順鉑單獨(dú)或聯(lián)合給藥,計(jì)算各組移植瘤的體積并繪制生長(zhǎng)曲線。給藥完成后,取移植瘤組織,免疫組化法檢測(cè)各組移植瘤組織Ki-67分子的表達(dá)水平,MSP技術(shù)檢測(cè)SOX2基因的甲基化情況,RT-PCR、Western blot分別檢測(cè)SOX2基因的mRNA和蛋白表達(dá)水平。結(jié)果:在體外細(xì)胞實(shí)驗(yàn)中,地西他濱(5μM)、順鉑(1μM、10μM、100μM)以及兩者的聯(lián)合應(yīng)用均能以時(shí)間和(或)劑量依賴性的方式抑制人胃癌細(xì)胞BGC-823的生長(zhǎng)增殖和遷移能力,均能一定程度地引起SOX2基因啟動(dòng)子區(qū)域DNA的去甲基化,增加SOX2基因的m RNA和蛋白表達(dá)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(duì)移植瘤的生長(zhǎng)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(zhǎng)和增殖能力。類(lèi)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(diào)了SOX2基因的mRNA和蛋白表達(dá)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(jié)論:在體外細(xì)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(zhuǎn)SOX2基因的甲基化狀態(tài),重新恢復(fù)其mRNA和蛋白的表達(dá),從而在抑制細(xì)胞生長(zhǎng)增殖和遷移、誘導(dǎo)細(xì)胞凋亡、抑制移植瘤的生長(zhǎng)和惡性增殖能力等過(guò)程中協(xié)同發(fā)揮抗腫瘤效應(yīng)。
[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(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R735.2

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