人剪切修复基因XPD抑制肝癌HepG2细胞增殖迁移及对自噬水平调控的实验研究

发布时间：2018-01-07 03:01

本文关键词：人剪切修复基因XPD抑制肝癌HepG2细胞增殖迁移及对自噬水平调控的实验研究　出处：《南昌大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的:探究上调肝癌细胞株HepG2中XPD基因的表达后对肿瘤细胞增殖凋亡、迁移以及自噬水平变化的影响。方法:采用脂质体作为载体介质将重组质粒pEGFP-N2/XPD和空载质粒pEGFP-N2瞬时转染入人肝癌细胞株HepG2细胞内,实验分为四组1)空白对照组、2)脂质体组、3)pEGFP-N2组、4)pEGFP-N2/XPD组,使用荧光显微镜观察绿色荧光表达情况;MTT法观察肿瘤细胞的增殖能力;流式细胞仪检测肿瘤细胞凋亡情况;体外平板克隆形成实验观察肿瘤细胞体外克隆集落形成情况;划痕愈合实验及Transwell实验检测肿瘤细胞迁移能力的变化。Western blotting检测HepG2细胞XPD及自噬相关蛋白LC3、P62表达量的变化。结果:荧光显微镜观察转染空载质粒pEGFP-N2和重组质粒pEGFPN2/XPD的细胞可见绿色荧光,转染有效。上调XPD的表达后,结果显示与对照组比较,实验组细胞增殖能力减弱(p0.01),细胞凋亡率增加(p0.01),体外克隆形成数量减少(p0.01),迁移能力减弱(p0.05),并且上调LC3蛋白表达(p0.01),而随之P62的表达水平减少(p0.01)。结论:过表达XPD基因能显著抑制肝癌细胞增殖促进其凋亡,抑制体外克隆形成,并且降低肿瘤细胞的迁移能力,同时调控肝癌细胞自噬水平的活化。
[Abstract]:Objective: to investigate the proliferation and apoptosis of hepatoma cells after up-regulating the expression of XPD gene in hepatoma cell line HepG2. Effects of migration and changes in autophagy levels. Methods:. The recombinant plasmid pEGFP-N2/XPD and the empty plasmid pEGFP-N2 were transiently transfected into human hepatoma cell line HepG2 cells using liposome as carrier medium. The experiment was divided into four groups (1) blank control group (2)) liposome group (pEGFP-N2) and pEGFP-N _ 2 / XPD group. The expression of green fluorescence was observed by fluorescence microscope. The proliferative ability of tumor cells was observed by MTT method. Apoptosis of tumor cells was detected by flow cytometry. The colony formation of tumor cells in vitro was observed by plate clone formation in vitro. Effect of scratch healing test and Transwell assay on the migration ability of tumor cells. XPD and autophagy associated protein LC3 in HepG2 cells were detected by blotting. Results: green fluorescence was observed in the cells transfected with empty plasmid pEGFP-N2 and recombinant plasmid pEGFPN2/XPD. After up-regulating the expression of XPD, the results showed that compared with the control group, the proliferation ability of the experimental group decreased p0.01a, and the apoptosis rate increased (p0.01). The number of clone formation in vitro decreased p0.01a, the migration capacity decreased, and the expression of LC3 protein was upregulated (p0.01). Conclusion: overexpression of XPD gene can significantly inhibit the proliferation of hepatoma cells and promote its apoptosis, and inhibit the formation of in vitro cloning. It also reduces the migration ability of tumor cells and regulates the activation of autophagy of hepatoma cells.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.7

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