TAB182在电离辐射诱导HeLa细胞DNA损伤修复中的作用

发布时间：2018-05-28 15:21

本文选题：TAB182 + DNA双链断裂　；参考：《中南大学》2012年硕士论文

【摘要】：目的研究TAB182参与电离辐射诱导DNA损伤修复的生物学功能,初步探讨TAB182参与DNA损伤修复的分子机制,进一步从分子水平上认识人类的健康和疾病发生的基础,为维持人类健康和疾病治疗药物特别是抗癌药物的研发提供分子水平的理论依据。实验方法通过shRNA技术构建TAB182基因沉默的细胞株,研究TAB182基因沉默的细胞株在电离辐射诱导DNA损伤的实验条件下,平板克隆形成实验研究TAB182基因沉默的细胞株的放射敏感性,流式细胞术分析电离辐射诱导的TAB182基因沉默细胞株的细胞周期阻滞,Western blot分析TAB182韶基因沉默的细胞中DNA损伤修复相关蛋白的水平。TDR双阻滞法对TAB182基因沉默的细胞进行细胞周期同步化,研究TAB182在细胞周期进程中的作用。HeLa细胞中瞬时转染PLPC-Myc-TAB182-FL载体,Western blot和激光共聚焦显微镜分析TAB182对DNA-PKcs磷酸化的影响,通过免疫共沉淀技术研究内源性的TAB182与DNA-PKcs的相互作用以及TNKS与DNA-PKcs的相互作用。结果 1.TAB182受电离辐射诱导表达。HeLa细胞在经过4Gy照射处理后,TAB182在2h左右表达水平最高；HeLa细胞经过不同剂量的电离辐射处理,2h后HeLa细胞中TAB182的表达含量经过4Gy照射剂量处理组表达最高,说明TAB182主要受中低剂量的电离辐射诱导表达,且TAB182的表达存在一定剂量依赖性。 2.TAB182、DNA-PKcs、TNKS的相互作用。免疫荧光原位杂交试验发现TAB182与2056位点磷酸化的DNA-PKcs存在共定位,免疫共沉淀证实TAB182与DNA-Pkcs存在相互作用,TNKS与DNA-Pkcs也存在相互作用。 3. shRNA沉默TAB182表达导致HeLa细胞放射敏感性增加。通过shRNA技术建立了TAB182基因稳定沉默的细胞株,通过细胞克隆形成实验发现TAB182基因沉默的细胞株对4Gy内的中低剂量γ射线照射的敏感性明显增加,但并不增加对8Gy大剂量照射的敏感性。 4.TAB182参与细胞周期进程的调节。Tdr双阻滞法同步细胞周期发现TAB182在细胞不同的细胞周期进程中表达含量不同,同时同步化TAB182基因沉默的细胞的细胞周期发现,与对照细胞相比,TAB182基因沉默的细胞S期进程加快,说明TAB182参与细胞周期S期进程的调节。 5.TAB182参与电离辐射诱导的细胞周期阻滞进程的调节。流式细胞术分析电离辐射诱导的细胞周期阻滞发现,TAB182基因沉默的细胞G2/M期阻滞时间明显比对照细胞长。 6.TABl82对DNA损伤应激相关蛋白表达的调节。Western Blot分析发现在TAB182基因沉默的细胞中DNA损伤修复蛋白如DNA-Pkcs、ATM、Chk2、P53的水平与对照细胞相比明显要低。 7.TAB182对DNA-PKcs磷酸化的影响。在HeLa细胞中瞬时转染PLPC-myc-TAB182-FL质粒,Western blot和免疫荧光显微镜实验分析表明TAB182能够增加DNA-PKcs自磷酸化位点S2056的磷酸化,Western blot分析TAB182基因沉默的细胞中DNA-Pkcs各磷酸化位点的磷酸化也得到一致的结果。结论 1.TAB182参与DNA损伤修复通路, 2.TAB182与DNA-PKcs存在相互作用,能够特异性的增加DNA-PKcs的自磷酸化位点S2056的磷酸化, 3.TAB182参与有丝分裂S期进程的调节和电离辐射诱导G2/M期阻滞进程的调节。
[Abstract]:objective
Study the biological function of TAB182 involved in DNA damage repair induced by ionizing radiation, preliminarily explore the molecular mechanism of TAB182 involvement in the repair of DNA damage, and further understand the basis of human health and disease from the molecular level, and provide a molecular level theory for the development of human health and disease treatment drugs, especially anticancer drugs. Basis.
Experimental method
The cell line of TAB182 gene silencing was constructed by shRNA technique, and the cell lines of the TAB182 gene silenced in the experimental conditions of DNA damage induced by ionizing radiation were studied. The plate clone formed the radiosensitivity of the cell lines with the TAB182 gene silencing, and the flow cytometry was used to analyze the cell cycle of the TAB182 gene silencing cell lines induced by ionizing radiation. Phase block, Western blot analysis of DNA damage and repair related proteins in TAB182 Shao gene silencing cells,.TDR double block method to synchronize cell cycle of TAB182 gene silenced cells, study the role of TAB182 in the process of cell cycle and transient transfection of PLPC-Myc-TAB182-FL carrier in.HeLa cells, Western blot and laser confocal The effect of TAB182 on the phosphorylation of DNA-PKcs was analyzed by microscope, and the interaction between endogenous TAB182 and DNA-PKcs and the interaction between TNKS and DNA-PKcs was studied by immunoprecipitation.
Result
The expression level of TAB182 was highest at 2h after 4Gy irradiation induced by ionizing radiation in 1.TAB182. HeLa cells were treated with different doses of ionizing radiation. The expression of TAB182 in HeLa cells after 2H was highest after 4Gy irradiated dose treatment group. It was said that TAB182 mainly was induced by low dose ionizing radiation. The expression of TAB182 was in a dose-dependent manner.
The interaction of 2.TAB182, DNA-PKcs and TNKS. Immunofluorescence in situ hybridization showed that TAB182 was Co located with DNA-PKcs of phosphorylation of 2056 loci. The interaction between TAB182 and DNA-Pkcs was confirmed by immunoprecipitation, and there was also interaction between TNKS and DNA-Pkcs.
3. shRNA silent TAB182 expression led to increased radiosensitivity in HeLa cells. A cell line that was stable and silent by TAB182 gene was established by shRNA technique. By cell clone formation experiments, the sensitivity of TAB182 gene silenced cell lines to low dose gamma ray irradiation in 4Gy was significantly increased, but it did not increase the sensitivity to large doses of 8Gy. Sensibility.
4.TAB182 participates in the cell cycle process and regulates the.Tdr double block method to synchronize the cell cycle. It is found that the expression of TAB182 in the cell cycle process is different, and the cell cycle of the cells that synchronize the TAB182 gene silencing is found. Compared with the control cells, the S stage of the TAB182 gene silencing cells is accelerated, indicating that TAB182 participates in the cells. The regulation of the period S period.
5.TAB182 participates in the regulation of the process of cell cycle arrest induced by ionizing radiation. Flow cytometry analysis of cell cycle arrest induced by ionizing radiation shows that the G2/M phase arrest time of TAB182 gene silencing cells is significantly longer than that of the control cells.
The regulation of DNA damage stress related protein expression by 6.TABl82.Western Blot analysis found that the level of DNA damage repair proteins such as DNA-Pkcs, ATM, Chk2, P53 in the cells with TAB182 gene silencing was significantly lower than that of the control cells.
The effect of 7.TAB182 on the phosphorylation of DNA-PKcs. PLPC-myc-TAB182-FL plasmids were transiently transfected in HeLa cells. Western blot and immunofluorescence microscopy showed that TAB182 could increase the phosphorylation of S2056 at the DNA-PKcs autophosphorylation site. Western blot analyzed the phosphorylation of each phosphorylation site in the cell of the TAB182 gene silencing. To a consistent result.
conclusion
1.TAB182 participates in the DNA damage repair pathway,
The interaction between 2.TAB182 and DNA-PKcs can specifically increase the phosphorylation of S2056 at DNA-PKcs self phosphorylation site.
3.TAB182 participates in the regulation of S phase in mitosis and the regulation of G2/M phase arrest process induced by ionizing radiation.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R114

【共引文献】