發(fā)布時(shí)間：2018-05-28 15:21

  本文選題：TAB182 + DNA雙鏈斷裂�。� 參考：《中南大學(xué)》2012年碩士論文

【摘要】：目的 研究TAB182參與電離輻射誘導(dǎo)DNA損傷修復(fù)的生物學(xué)功能,初步探討TAB182參與DNA損傷修復(fù)的分子機(jī)制,進(jìn)一步從分子水平上認(rèn)識(shí)人類的健康和疾病發(fā)生的基礎(chǔ),為維持人類健康和疾病治療藥物特別是抗癌藥物的研發(fā)提供分子水平的理論依據(jù)。 實(shí)驗(yàn)方法 通過shRNA技術(shù)構(gòu)建TAB182基因沉默的細(xì)胞株,研究TAB182基因沉默的細(xì)胞株在電離輻射誘導(dǎo)DNA損傷的實(shí)驗(yàn)條件下,平板克隆形成實(shí)驗(yàn)研究TAB182基因沉默的細(xì)胞株的放射敏感性,流式細(xì)胞術(shù)分析電離輻射誘導(dǎo)的TAB182基因沉默細(xì)胞株的細(xì)胞周期阻滯,Western blot分析TAB182韶基因沉默的細(xì)胞中DNA損傷修復(fù)相關(guān)蛋白的水平。TDR雙阻滯法對(duì)TAB182基因沉默的細(xì)胞進(jìn)行細(xì)胞周期同步化,研究TAB182在細(xì)胞周期進(jìn)程中的作用。HeLa細(xì)胞中瞬時(shí)轉(zhuǎn)染PLPC-Myc-TAB182-FL載體,Western blot和激光共聚焦顯微鏡分析TAB182對(duì)DNA-PKcs磷酸化的影響,通過免疫共沉淀技術(shù)研究?jī)?nèi)源性的TAB182與DNA-PKcs的相互作用以及TNKS與DNA-PKcs的相互作用。 結(jié)果 1.TAB182受電離輻射誘導(dǎo)表達(dá)。HeLa細(xì)胞在經(jīng)過4Gy照射處理后,TAB182在2h左右表達(dá)水平最高；HeLa細(xì)胞經(jīng)過不同劑量的電離輻射處理,2h后HeLa細(xì)胞中TAB182的表達(dá)含量經(jīng)過4Gy照射劑量處理組表達(dá)最高,說明TAB182主要受中低劑量的電離輻射誘導(dǎo)表達(dá),且TAB182的表達(dá)存在一定劑量依賴性。 2.TAB182、DNA-PKcs、TNKS的相互作用。免疫熒光原位雜交試驗(yàn)發(fā)現(xiàn)TAB182與2056位點(diǎn)磷酸化的DNA-PKcs存在共定位,免疫共沉淀證實(shí)TAB182與DNA-Pkcs存在相互作用,TNKS與DNA-Pkcs也存在相互作用。 3. shRNA沉默TAB182表達(dá)導(dǎo)致HeLa細(xì)胞放射敏感性增加。通過shRNA技術(shù)建立了TAB182基因穩(wěn)定沉默的細(xì)胞株,通過細(xì)胞克隆形成實(shí)驗(yàn)發(fā)現(xiàn)TAB182基因沉默的細(xì)胞株對(duì)4Gy內(nèi)的中低劑量γ射線照射的敏感性明顯增加,但并不增加對(duì)8Gy大劑量照射的敏感性。 4.TAB182參與細(xì)胞周期進(jìn)程的調(diào)節(jié)。Tdr雙阻滯法同步細(xì)胞周期發(fā)現(xiàn)TAB182在細(xì)胞不同的細(xì)胞周期進(jìn)程中表達(dá)含量不同,同時(shí)同步化TAB182基因沉默的細(xì)胞的細(xì)胞周期發(fā)現(xiàn),與對(duì)照細(xì)胞相比,TAB182基因沉默的細(xì)胞S期進(jìn)程加快,說明TAB182參與細(xì)胞周期S期進(jìn)程的調(diào)節(jié)。 5.TAB182參與電離輻射誘導(dǎo)的細(xì)胞周期阻滯進(jìn)程的調(diào)節(jié)。流式細(xì)胞術(shù)分析電離輻射誘導(dǎo)的細(xì)胞周期阻滯發(fā)現(xiàn),TAB182基因沉默的細(xì)胞G2/M期阻滯時(shí)間明顯比對(duì)照細(xì)胞長(zhǎng)。 6.TABl82對(duì)DNA損傷應(yīng)激相關(guān)蛋白表達(dá)的調(diào)節(jié)。Western Blot分析發(fā)現(xiàn)在TAB182基因沉默的細(xì)胞中DNA損傷修復(fù)蛋白如DNA-Pkcs、ATM、Chk2、P53的水平與對(duì)照細(xì)胞相比明顯要低。 7.TAB182對(duì)DNA-PKcs磷酸化的影響。在HeLa細(xì)胞中瞬時(shí)轉(zhuǎn)染PLPC-myc-TAB182-FL質(zhì)粒,Western blot和免疫熒光顯微鏡實(shí)驗(yàn)分析表明TAB182能夠增加DNA-PKcs自磷酸化位點(diǎn)S2056的磷酸化,Western blot分析TAB182基因沉默的細(xì)胞中DNA-Pkcs各磷酸化位點(diǎn)的磷酸化也得到一致的結(jié)果。 結(jié)論 1.TAB182參與DNA損傷修復(fù)通路, 2.TAB182與DNA-PKcs存在相互作用,能夠特異性的增加DNA-PKcs的自磷酸化位點(diǎn)S2056的磷酸化, 3.TAB182參與有絲分裂S期進(jìn)程的調(diào)節(jié)和電離輻射誘導(dǎo)G2/M期阻滯進(jìn)程的調(diào)節(jié)。
[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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R114

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