【作者】 徐勇；

【导师】 周晓中；

【作者基本信息】 苏州大学 ， 外科学， 2015， 硕士

【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(*p<0.05)。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(*p<0.05);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(*p<0.05),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(*p<0.05);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(*p<0.05);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。更多还原

【Abstract】 Objective: To investigate the activity of AKT signaling pathway after LDI and elucidate the underlying mechanism of LDI-induced osteoblast differentiation.Methods:1. Osteoblasts were harvested from the calvariae of 2-or 3-day old mice and exposed to single dose of X- irradiation with 0.0 Gy、0.5 Gy、1.0 Gy、2.0 Gy and 4.0 Gy respectively. The cell viability was measured by the 3- [4,5-dim-ethylthylthiazol-2- yl]-2,5 diphenyltetrazolium bromide(MTT) assay. Alkaline Phosphate(ALP) activity was evaluated by ALP viability kit. The expression of type I collagen(Col I) and Runt-related transcription factor-2(Runx2) were measured by Real- time PCR.2. Osteoblasts were exposed to single dose of X- irradiation with 0.0 Gy and 1.0 Gy. The activity of AKT was measured by Western-blot. After using the kinase inhibitors against Akt or DNA-PKcs, as well as the sh RN A partial depletion of Akt1, Alkaline Phosphate(ALP) activity was evaluated by ALP viability kit. The expression of type I collagen(Col I)and Runt- related transcription factor 2(Runx2) were measured by Realtime PCR. The target of LDI- induced AKT activity was explored by immunoprecipitation(Co-IP) assay.Results : Results from MTT assay demonstrated that only 4 Gy X-ray irradiation reduced cell viability of mouse calvarial osteoblasts, no significant effect were showed on osteoblast survival at 0.0 Gy、0.5 Gy、1.0 Gy、2.0 Gy(*p < 0.05). X-ray irradiation from 0.5 Gy to 2 Gy significantly increased ALP activity in osteoblasts, while X-ray irradiation at a high dose(4 Gy) failed to do so(*p < 0.05). X-ray irradiation from 0.5 Gy to 2 Gy induced m RNA expression of Col I and Runx2(*p < 0.05). LDI at 1 Gy showed most significant effect on osteoblast differentiation. DNA-PKcs activation is required for LDI- induced ALP activity increase and Col I/Runx2 m RNA expression in osteoblasts(*p < 0.05). Akt activation is involved in LDI- induced osteoblast differentiation(*p < 0.05). Co-IP assay results showed that LDI induced DNA-PKcs-SIN1 complexation in mouse calvarial osteoblasts(*p < 0.05), which was inhibited by NU- 7441 or by SIN1 si RNA-knockdown.Conclusion: DNA-PKcs-SIN1 complexation is required for LDI- induced Akt activation and mouse calvarial osteoblast differentiation.更多还原