【作者】 罗加林；

【导师】 曹建平；

【作者基本信息】 苏州大学 ， 放射医学， 2005， 硕士

【摘要】 目的:利用胞质分裂阻滞微核法、常规染色体畸变分析技术及HPRT 基因突变分析技术,分别从细胞、染色体和分子水平整体研究AT 细胞辐射敏感性;利用基因转染技术构建PEBS7-YZ5 稳定表达AT 细胞株,研究外源性ATM 蛋白对P53 蛋白的调控;以K562 细胞为P53 突变细胞模型,研究ATM 蛋白对P21 蛋白的直接调控,从而从信号传导通路角度,探讨AT 细胞高辐射敏感性的机制。方法:(1)以源于正常人皮肤的成纤维细胞系GM0639(GM 细胞)为对照,采用胞质分裂阻滞微核法,在AT 细胞和GM 细胞经(60)~Coγ射线0、1、2、3、4Gy 照射后,观察比较AT 细胞和GM 细胞之间微核分布、微核率及微核细胞率的差异;(2)以GM 细胞为对照,用常规染色体畸变分析法,在AT5BIVA(AT 细胞)和GM 细胞经(60)~Coγ射线0、1、2、3、4Gy 照射后,观察比较AT 细胞和GM 细胞之间染色体畸变率的差异;(3)以GM 细胞为对照,利用次黄嘌呤磷酸核糖转移酶基因位点突变分析技术,在AT 细胞和GM 细胞经(60)~Coγ射线0、1、2、3、4Gy 照射后,观察比较AT 细胞和GM 细胞之间hprt 基因位点突变频率的差异;(4)利用电穿孔技术,将含有ATM 基因cDNA 的真核表达载体PEBS7-YZ5 转染到AT 细胞中,用潮霉素筛选以获得稳定表达细胞株,RT-PCR 检测ATM mRNA 的转录,Western blot进一步验证ATM 蛋白的表达;(5)利用免疫共沉淀及Western blot 技术研究在PEBS7-YZ5-AT 稳定转染细胞株中,表达的ATM 蛋白是否调控P53 蛋白的磷酸化,以明确ATM 基因与p53 的关系;(6)以p53 突变的K562 细胞为模型,利用免疫共沉淀及Western blot 技术研究ATM 蛋白与P21 蛋白的直接相互作用,探讨ATM 是否不通过P53 而直接调控P21 的磷酸化。结果:(1)经1、2、3、4Gy(60)~Coγ射线照射后,在同一剂量点,AT 细胞单核、多核微核细胞率、微核率及微核细胞率均明显高于GM 细胞(P<0.01),并且两者微核率及微核细胞率均与剂量呈正相关,均可拟合成剂量效应直线方程y=a+bx,AT 细胞微核率及微核细胞率直线回归方程斜率明显大于GM 细胞(P<0.01);(2)在同一实验剂量下,AT 细胞染色体畸变率明显高于GM 细胞(P<0.05),AT 和GM 细胞染色更多还原

【Abstract】 Objective: Cytokinesis-Block micronucleus method, Conventional chromosome aberraton analysis method and Hypoxanthine phospho-ribosyl transferase gene mutation analysis technique were used to study the radiosensitivity of Ataxia-telangiectasia fibroblast cells at the cell, chromosome and molecular level respectively. Using gene transfection technique,we established the AT cell strains with PEBS7-YZ5 plasmid in which we aimed to study whether ATM controlled the phosphorylation of P53. K562 cells served as a p53 mutation model in which we intended to study whether ATM controlled the phosphorylation of P21 directly without the mediation of P53. Accordingly we study the mechanism of the high radiosensitivity of AT cell from the aspect of signal transduction pathway Methods: (1)Using Cytokinesis-Block micronucleus method, the micronucleus frequencies(MNF) and micronucleus cell frequencies(MNCF) of AT cells (AT5BIVA) with 0, 1, 2, 3 and 4 Gy exposure to (60)~Co γ-rays were observed, compared with those of GM cells (GM0639) from the skin of the normal subject; (2)Using conventional chromosome aberration analysis method, chromosome aberration frequences(CAF) of AT cells with 0,1,2,3 and 4 Gy exposure to (60)~Co γ-rays were observed, compared with that of GM cells; (3)Using Hypoxanthine phospho-ribosyl transferase gene mutation analysis technique, the hprt mutation frequencies (hprtMF) of AT cells irradiated with 0, 1, 2, 3 and 4 Gy of 60Co γ-rays were observed, compared with that of GM cells; (4)PEBS7-YZ5 plasmids containing ATM gene cDNA were transfected into AT cells by electroporation. Hygromicin is used to select the cells expressing ATM protein stably. RT-PCR was used to detect transcription of ATM and Western blot was used to detect expression of ATM protein in order to verify the ATM gene transfection. (5)The Co-immunoprecipitation and Western blot technique were used to study whether ATM controlled the phosphorylation of P53 and identify the interrelationship between ATM and p53 in PEBS7-YZ5-AT cells. (6)K562 cells serve as a p53 mutation model in which we used co-immunoprecipitation and Western blot technique to study the interrelationship between ATM and p21, and identify whether ATM phosphorylated p21 directly without the mediation of P53. Results: (1)After exposed to 1, 2, 3 and 4 Gy of 60Co γ-rays, the MNF and MNCF induced by irradiation were significantly higher in the AT cells compared with those of GM cells(P<0.01). In the two cell types, both MNF and MNCF had a positive correlation with dose, and the linear regression equations of AT and GM cells were: Y=a+bX. The slopes of MNF and MNCF linear regression equations in AT cells are larger than those of GM(P<0.01) ; (2)After exposed to 0, 1, 2, 3 and 4 Gy of 60Co γ-rays, the CAF induced by irradiation was significantly higher in the AT cells compared with that of GM cells(P<0.05). In the two cell types, CAF had a positive correlation with dose, and the linear regression equations of AT and GM cells were Y=a+bX. The slope of CAF linear regression equations of AT cells is larger than that of GM cells(P<0.05); (3)After exposure to 0, 1, 2, 3 and 4 Gy of 60Co γ-rays, hprt MF was significantly higher in AT cells than that in GM cells(P<0.01). In the two cell types, hprt MF had a positive correlation with dose, and their linear regression equations were y=a+bx. The slope of AT hprt MF linear regression equations were larger than that of GM(P<0.05); (4)PEBS7-YZ5 plasmids were transfected into AT cells successfully. Cell strains expressing ATM protein stably were obtained after selected by hygromicin. RT-PCR detected fragment of ATM cDNA. Western blot detected blot of ATM protein; (5)After exposed to ionizing radiation, P53 of PEBS7-YZ5-AT cells was phosphorylated. Immunoprecipitation showed that ATM interreacted with p53; (6)P21 of K562 cells was phosphorylated after exposure to 60Co γ-rays. P21 protein was found in the Immunoprecipitation complex by ATM antibody. Conclusion: (1)After exposed to 1, 2, 3 and 4 Gy of 60Co γ-rays, both MNF and MNCF of AT and GM cells had a positive correlation with dose. At the same dose point, the MNF and MNCF of AT cells were significantly higher than those of GM cells (P<0.01). So we concluded that the radiosensitivity of AT cells is significantly higher than that of GM cells at the cell level;(2) After exposed to 0, 1, 2, 3 and 4 Gy of 60Co γ-rays, CAF of AT and GM cells had a positive correlation with dose. At the same dose point, the CAF of AT cells was significantly higher than that of GM cells(P < 0.01). It showed that theradiation-induced chromosome damage of AT cells is more serious than that of GM cells and the radiosensitivity of AT cells is significantly higher than that of GM cells; (3) After exposed to 0, 1, 2, 3 and 4 Gy of 60Co γ-rays, hprt MF of AT and GM cells had a positive correlation with dose. At the same dose point, the hprt MF of AT cells was significantly higher than that of GM cells(P<0.01). It showed that the radiation-induced genetic locus damage of AT cells is more serious than that of GM cells, and the radiosensitivity of AT cells is significantly higher than that of GM cells; (4)In the AT cell strains transfected with PEBS7-YZ5 plasmid, exogenous ATM mRNA and ATM protein expressed stably; (5)Ionizing radiation can activate exogenous ATM kinase, and activated ATM kinase phosphorylated downstream p53 gene. More P53 was phosphorylated by ATM with the increase of radiation dose;(6) Radiation-induced ATM kinase can phosphorylate P21 directly without P53 mediation. And more P21 was phosphorylated by ATM with the radiation dose increasing.更多还原