本文選題：低劑量X射線　切入點(diǎn)：AKT信號(hào)通路　出處：《蘇州大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:觀察低劑量X射線作用于成骨細(xì)胞后AKT的活化情況,并探討低劑量X射線促進(jìn)成骨細(xì)胞分化的機(jī)制。方法:1、提取小鼠成骨細(xì)胞,分別給予單次劑量0.0 Gy、0.5 Gy、1.0 Gy、2.0 Gy和4.0 Gy照射。運(yùn)用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴鹽(MTT)檢測(cè)細(xì)胞增殖;ALP試劑盒檢測(cè)細(xì)胞早期分化情況;Real-time PCR檢測(cè)I型膠原蛋白(Col-I)和成骨特異性轉(zhuǎn)錄因子2(Runx2)的表達(dá)。2、小鼠成骨細(xì)胞分別給予單次劑量0.0 Gy、1.0 Gy照射。運(yùn)用Western-blot法測(cè)定AKT信號(hào)通路相關(guān)蛋白的活化;分別應(yīng)用信號(hào)通路抑制劑、基因沉默(RNA i)等方法阻斷AKT信號(hào)通路后,使用ALP試劑盒檢測(cè)細(xì)胞早期分化情況及Real-time PCR檢測(cè)Col-1和Runx2的表達(dá);利用免疫共沉淀法(Co-IP)測(cè)定DNA-PKcs-SIN1復(fù)合物的存在,探測(cè)低劑量射線作用于AKT信號(hào)通路的靶點(diǎn)。結(jié)果:1、單次劑量照射72 h后,細(xì)胞生存活性在0.0 Gy、0.5 Gy、1.0 Gy、2.0 Gy各組間無明顯差異,而4.0 Gy照射組細(xì)胞生存活性明顯下降(*p0.05)。1.0 Gy射線照射組ALP活性水平、Col-I的表達(dá)及Runx2的表達(dá)高于其它各組(*p0.05);2、Western-bolt結(jié)果顯示1.0 Gy射線照射組較對(duì)照組(0.0 Gy)明顯激活A(yù)KT Ser-473位點(diǎn)(*p0.05),且1.0 Gy射線照射組DNA-PKcs Thr2647位點(diǎn)及Thr2609位點(diǎn)叫對(duì)照組明顯磷酸化(*p0.05);使用通路阻斷劑或RNA i等,明顯抑制了低劑量X射線激活A(yù)KT信號(hào)通路的作用,并抑制了低劑量X射線所促進(jìn)的ALP活性、Col-I的表達(dá)及Runx2的表達(dá)的作用(*p0.05);免疫共沉淀結(jié)果證實(shí)了DNA-PKcs-SIN1復(fù)合物的存在,且該復(fù)合物可以被信號(hào)通路抑制劑和RNAi抑制。(*p.05)。結(jié)論:低劑量X射線依賴DNA-PKcs-SIN1復(fù)合物促進(jìn)AKT信號(hào)通路活化和成骨細(xì)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.

【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R683

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相關(guān)期刊論文 前1條

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本文編號(hào)：1570059