慢病毒介导Period2低表达促进X线照射胶质瘤U343细胞凋亡的分子机制研究

发布时间：2018-04-02 06:24

本文选题：Per2　切入点：U343　出处：《宁夏医科大学》2014年硕士论文

【摘要】：目的体外研究生物钟基因Period2（Per2）的低表达对X线照射引起的胶质瘤细胞凋亡的影响及其分子机制。方法构建并合成Per2-shRNA、Control-shRNA（NC）慢病毒，将其感染p53野生型人胶质瘤U343细胞，应用嘌呤霉素筛选Per2低表达的shRNA-Per2细胞组及Control对照细胞组；给予6mV100cGy X线照射两组实验细胞，并分别于1h、12h、24h后，应用单细胞凝胶电泳技术检测两组U343细胞DNA的损伤情况、应用Annexin V-FITC/PI流式细胞术检测实验组细胞的凋亡情况，，并应用RT-qPCR及Western Blot技术检测X线照射后两组U343细胞中与DNA损伤修复、细胞凋亡相关的基因ATM、p53以及c-myc等的表达情况。结果Per2-shRNA慢病毒明显下调生物钟基因Per2在人胶质瘤U343细胞中的表达；给予X线照射后，与Control细胞组相比，Per2低表达的shRNA-Per2细胞组DNA损伤更为严重，细胞凋亡率明显增加，且在X线照射后的1h、12h、24h，随着时间变化，shRNA-Per2细胞组的DNA损伤和细胞凋亡呈现出递增的趋势；此外，随Per2蛋白表达量的下调，ATM和p53的表达水平也明显减少，而c-myc的表达水平显著增加。结论下调生物钟基因Per2的表达可以增加人胶质瘤U343细胞对X线的敏感性；且Per2作为上游基因，通过调控ATM、p53、c-myc等的表达，减弱了它们在ATM-p53通路中对损伤DNA的修复作用，进一步促进了胶质瘤细胞U343的凋亡；这一研究也为后期临床靶向基因治疗胶质瘤打下了理论基础。
[Abstract]:Objective to investigate the biological clock gene Period2 (Per2) low expression caused by X-ray irradiation of glioma cell apoptosis effect and its molecular mechanism.
The construction method and the synthesis of Per2-shRNA, Control-shRNA (NC) lentivirus, the infection of wild type p53 in human glioma U343 cells, using puromycin screening shRNA-Per2 cell group and the low expression of Control Per2 cell control group;
Given the 6mV100cGy X-ray irradiation two groups of experimental cells, and respectively in 1H, 12h, 24h after injury was detected by single cell gel electrophoresis two U343 cell DNA, apoptosis by Annexin V-FITC/PI flow cytometry cells in the experimental group, and the application of X-ray exposure and RT-qPCR Western Blot of U343 cells after two in the group with DNA damage repair, apoptosis related gene expression of ATM, p53 and c-myc.
Results Per2-shRNA expression of lentiviral down-regulation of clock gene Per2 in human glioma U343 cells; given after X-ray irradiation, compared with Control cells group, shRNA-Per2 cells group DNA damage to the low expression of Per2 is more serious, the apoptosis rate was significantly increased, and in the X-ray irradiation of 1H, 12h, 24h, along with the time change, DNA damage and cell apoptosis of shRNA-Per2 group showed a trend of increasing; in addition, with the expression of Per2 protein was down regulated, the expression level of ATM and p53 decreased significantly, while the expression level of c-myc was significantly increased.
Conclusion the expression down-regulation of clock gene Per2 can increase the sensitivity of human glioma U343 cells to X-ray; and Per2 as the upstream gene regulated by ATM, p53, and the expression of c-myc, weakened them in the ATM-p53 pathway of DNA damage repair effect, to further promote the apoptosis of glioma cells U343; this study for later clinical targeted gene therapy of glioma lay a theoretical foundation.

【学位授予单位】：宁夏医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.41

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