DNA-PKcs-SIN1复合物介导的低剂量X射线促进AKT活化及成骨细胞分化

发布时间：2018-03-05 11:24

本文选题：低剂量X射线　切入点：AKT信号通路　出处：《苏州大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的:观察低剂量X射线作用于成骨细胞后AKT的活化情况,并探讨低剂量X射线促进成骨细胞分化的机制。方法:1、提取小鼠成骨细胞,分别给予单次剂量0.0 Gy、0.5 Gy、1.0 Gy、2.0 Gy和4.0 Gy照射。运用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)检测细胞增殖;ALP试剂盒检测细胞早期分化情况;Real-time PCR检测I型胶原蛋白(Col-I)和成骨特异性转录因子2(Runx2)的表达。2、小鼠成骨细胞分别给予单次剂量0.0 Gy、1.0 Gy照射。运用Western-blot法测定AKT信号通路相关蛋白的活化;分别应用信号通路抑制剂、基因沉默(RNA i)等方法阻断AKT信号通路后,使用ALP试剂盒检测细胞早期分化情况及Real-time PCR检测Col-1和Runx2的表达;利用免疫共沉淀法(Co-IP)测定DNA-PKcs-SIN1复合物的存在,探测低剂量射线作用于AKT信号通路的靶点。结果:1、单次剂量照射72 h后,细胞生存活性在0.0 Gy、0.5 Gy、1.0 Gy、2.0 Gy各组间无明显差异,而4.0 Gy照射组细胞生存活性明显下降(*p0.05)。1.0 Gy射线照射组ALP活性水平、Col-I的表达及Runx2的表达高于其它各组(*p0.05);2、Western-bolt结果显示1.0 Gy射线照射组较对照组(0.0 Gy)明显激活AKT Ser-473位点(*p0.05),且1.0 Gy射线照射组DNA-PKcs Thr2647位点及Thr2609位点叫对照组明显磷酸化(*p0.05);使用通路阻断剂或RNA i等,明显抑制了低剂量X射线激活AKT信号通路的作用,并抑制了低剂量X射线所促进的ALP活性、Col-I的表达及Runx2的表达的作用(*p0.05);免疫共沉淀结果证实了DNA-PKcs-SIN1复合物的存在,且该复合物可以被信号通路抑制剂和RNAi抑制。(*p.05)。结论:低剂量X射线依赖DNA-PKcs-SIN1复合物促进AKT信号通路活化和成骨细胞分化。
[Abstract]:Objective: To observe the effect of low dose X ray in the activation of AKT cells after bone, and to explore the mechanism of low dose X ray to promote bone cell differentiation. Methods: 1, extraction of mouse osteoblasts, were given a single dose of 0 Gy, 0.5 Gy, 1 Gy, 2 Gy and 4 Gy by irradiation. 3- (4,5- two -2 -2,5- two methylthiazole) phenyl tetrazole bromide (MTT) detection of cell proliferation; ALP kit for detecting cells in early differentiation; Real-time PCR detection of type I collagen (Col-I) and osteoblast specific transcription factor 2 (Runx2) expression of.2 in mouse osteoblasts were given a single dose of 0 Gy, 1 Gy irradiation. The activation of AKT signaling pathway related protein were determined by Western-blot method; used signal transduction inhibitors, gene silencing (RNA I) method after blocking the AKT pathway using ALP assay for cell differentiation and early detection of Col-1 Runx2 and Real-time PCR Expression; co precipitation method using DNA-PKcs-SIN1 (Co-IP) determination of immune complexes in the presence of target detection, low dose irradiation effects on AKT signal pathway. Results: 1, a single dose of 72 h after irradiation, cell survival activity at 0 Gy, 0.5 Gy, 1 Gy, Gy no significant difference between the 2 groups. While the 4 Gy irradiation group significantly decreased cell survival activity (*p0.05) of ALP group activity level of Gy ray.1.0, expression of Runx2 and Col-I was higher than other groups (*p0.05); 2, Western-bolt showed 1 Gy irradiation group than in the control group (0 Gy) was activated AKT Ser-473 loci (*p0.05), and 1 Gy irradiation group DNA-PKcs Thr2647 sites and Thr2609 sites is significantly phosphorylated (*p0.05); using I or RNA pathway inhibitor, inhibited the low dose X ray activation of AKT signaling pathway and inhibit the low dose of X radiation promoted the activity of ALP, Col-I The expression and role of Runx2 expression (*p0.05); the results confirmed that DNA-PKcs-SIN1 complexes in the presence of CO immunoprecipitation, and the complexes can be pathway inhibitors and inhibition of RNAi. (*p.05). Conclusion: low dose X ray DNA-PKcs-SIN1 dependent complexes to promote the activation of AKT signaling pathway and osteoblast differentiation.

【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R683

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