食管鳞癌细胞中Bad对Dad1基因表达调控机制的研究

发布时间：2018-05-04 00:24

本文选题：食管鳞癌 + 调控　；参考：《新疆医科大学》2017年硕士论文

【摘要】：目的:初步阐述Bad在食管鳞癌细胞中对Dad1表达的调控作用,了解上调和下调Bad对食管鳞癌细胞周期和凋亡的影响。方法:(1)培养人食管鳞癌ECA109和KYSE450细胞,构建Bad过表达载体重组质粒GV142-Bad,转染食管鳞癌细胞组作为Bad高表达组,转染空载体的细胞组作为阴性对照组,完全培养基处理的细胞组作为空白对照组。转染24h后,荧光倒置显微镜观察细胞中绿色荧光蛋白的表达情况,提取总RNA和蛋白并使用qPCR及Western blot检测Bad在基因转录和蛋白水平的表达来验证转染效率。同时检测转染细胞中Dad1在基因转录和蛋白水平的表达。GV142-Bad载体转染48 h后,使用流式细胞仪检测上调Bad的表达对细胞周期和凋亡的影响。(2)人工合成针对人Bad基因的si RNA(si RNA-Bad),转染食管鳞癌ECA109和KYSE450细胞,作为Bad的低表达组,转染阴性对照si RNA(negative-si RNA)的细胞组为阴性对照组,完全培养基处理的细胞组作为空白对照组。转染24h后,提取总RNA和蛋白,使用qPCR以及Western blot检测Bad在基因转录水平和蛋白水平的表达,验证转染效率。同时检测Dad1在基因转录和蛋白水平的表达,验证下调Bad表达是否调控了Dad1的表达。si RNA-Bad转染48 h后,使用流式细胞仪检测下调Bad表达对细胞凋亡的影响。结果:(1)成功构建Bad过表达载体GV142-Bad,GV142-Bad过表达重组质粒转染食管鳞癌细胞ECA109和KYSE450后可以上调Bad在基因转录和蛋白水平的表达。同时检测到Dad1在基因转录和蛋白水平表达被下调。(2)成功合成针对人Bad基因的si RNA,转染食管鳞癌ECA109和KYSE450细胞后能够下调Bad在基因转录和蛋白水平的表达。同时检测到Dad1在基因转录和蛋白水平的表达被上调。(3)上调Bad基因的表达后观察到ECA109细胞周期G2/M期被延长,同时S期有缩短趋势,但统计结果差异不显著(P0.05)。而KYSE450细胞周期阻滞于G2/M期并伴有S期缩短,差异有统计学意义(P0.05)。上调Bad基因的表达后发现两种细胞的凋亡比率显著增加,差异有统计学意义(P0.05)。下调Bad基因表达后两种细胞的凋亡率有下降趋势但不显著,三组之间差异无统计学意义(P0.05)。结论:促凋亡基因Bad可能调控抗凋亡基因Dad1的表达,两者之间可能存在负调控关系,Bad基因可能通过将癌细胞周期阻滞于G2/M并且促进其凋亡来,抑制其增殖。
[Abstract]:Aim: to investigate the role of Bad in the regulation of Dad1 expression in esophageal squamous cell carcinoma cells and to understand the effects of up-regulation and down-regulation of Bad on cell cycle and apoptosis in esophageal squamous cell carcinoma cells. Methods Human esophageal squamous cell carcinoma (ECA109) and KYSE450 cells were cultured. Bad overexpression vector GV142-Badwas constructed. The transfected esophageal squamous carcinoma cells group was used as the high expression group of Bad and the empty vector group was used as the negative control group. The cells treated with complete culture medium were used as blank control group. After 24 hours of transfection, the expression of green fluorescent protein (GFP) was observed by fluorescence inverted microscope, the total RNA and protein were extracted, and the expression of Bad at gene transcription and protein level was detected by qPCR and Western blot to verify the transfection efficiency. At the same time, the expression of Dad1 at gene transcription and protein level in transfected cells was detected 48 h after transfection with GV142-Bad vector. The effect of upregulation of Bad expression on cell cycle and apoptosis was detected by flow cytometry (FCM). Si RNA(si RNA-Badsite targeting human Bad gene was synthesized and transfected into ECA109 and KYSE450 cells of esophageal squamous cell carcinoma as the low expression group of Bad. The cells transfected with si RNA(negative-si of negative control group were negative control group, and the cell group treated with complete culture medium served as blank control group. After 24 hours of transfection, the total RNA and protein were extracted, and the expression of Bad at the level of gene transcription and protein was detected by qPCR and Western blot to verify the transfection efficiency. At the same time, the expression of Dad1 at gene transcription and protein level was detected to verify whether the down-regulation of Bad expression regulated the expression of Dad1. Si RNA-Bad was transfected for 48 h. The effect of down-regulation of Bad expression on apoptosis was detected by flow cytometry. Results Bad overexpression vector GV142-BadGV142-Bad was successfully constructed and transfected into esophageal squamous cell ECA109 and KYSE450, which could up-regulate the expression of Bad at gene transcription and protein levels. The siRNAs targeting human Bad gene were successfully synthesized and transfected into ECA109 and KYSE450 cells of esophageal squamous cell carcinoma, which could down-regulate the expression of Bad at gene transcription and protein level. At the same time, it was found that Dad1 up-regulated the expression of Bad gene at the level of gene transcription and protein. After upregulating the expression of Bad gene, it was observed that the G2 / M phase of ECA109 cell cycle was prolonged and the S phase was shortened, but there was no significant difference in the statistical results (P 0.05). The cell cycle of KYSE450 was arrested at G _ 2 / M phase with S phase shortening, and the difference was statistically significant (P < 0.05). After upregulating the expression of Bad gene, it was found that the apoptotic ratio of the two kinds of cells was significantly increased, and the difference was statistically significant (P 0.05). After down-regulation of Bad gene expression, the apoptosis rate of the two kinds of cells decreased, but not significantly. There was no significant difference between the three groups (P 0.05). Conclusion: the pro-apoptotic gene Bad may regulate the expression of anti-apoptotic gene Dad1, and there may be a negative regulatory relationship between the two genes, which may inhibit the proliferation of cancer cells by blocking the cell cycle at G _ 2 / M and promoting its apoptosis.
【学位授予单位】：新疆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.1

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