本文選題：Per2　切入點：U343　出處：《寧夏醫(yī)科大學》2014年碩士論文

【摘要】：目的體外研究生物鐘基因Period2（Per2）的低表達對X線照射引起的膠質瘤細胞凋亡的影響及其分子機制。 方法構建并合成Per2-shRNA、Control-shRNA（NC）慢病毒，將其感染p53野生型人膠質瘤U343細胞，應用嘌呤霉素篩選Per2低表達的shRNA-Per2細胞組及Control對照細胞組； 給予6mV100cGy X線照射兩組實驗細胞，并分別于1h、12h、24h后，應用單細胞凝膠電泳技術檢測兩組U343細胞DNA的損傷情況、應用Annexin V-FITC/PI流式細胞術檢測實驗組細胞的凋亡情況，，并應用RT-qPCR及Western Blot技術檢測X線照射后兩組U343細胞中與DNA損傷修復、細胞凋亡相關的基因ATM、p53以及c-myc等的表達情況。 結果Per2-shRNA慢病毒明顯下調生物鐘基因Per2在人膠質瘤U343細胞中的表達；給予X線照射后，與Control細胞組相比，Per2低表達的shRNA-Per2細胞組DNA損傷更為嚴重，細胞凋亡率明顯增加，且在X線照射后的1h、12h、24h，隨著時間變化，shRNA-Per2細胞組的DNA損傷和細胞凋亡呈現(xiàn)出遞增的趨勢；此外，隨Per2蛋白表達量的下調，ATM和p53的表達水平也明顯減少，而c-myc的表達水平顯著增加。 結論下調生物鐘基因Per2的表達可以增加人膠質瘤U343細胞對X線的敏感性；且Per2作為上游基因，通過調控ATM、p53、c-myc等的表達，減弱了它們在ATM-p53通路中對損傷DNA的修復作用，進一步促進了膠質瘤細胞U343的凋亡；這一研究也為后期臨床靶向基因治療膠質瘤打下了理論基礎。
[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.

【學位授予單位】：寧夏醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R739.41

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本文編號：1699138