mTOR及其下游信号通路在骨髓间充质干细胞氧化应激损伤中的变化及作用

发布时间：2018-09-14 14:56

【摘要】：目的研究小鼠骨髓间充质干细胞(mouse bone marrow stem cells,mBMSCs)在氧化应激损伤中哺乳动物雷帕霉素靶蛋白(mammaliatargetofrapamycin,mTOR)及其下游信号通路的变化及其作用。方法从30只雄性健康昆明小鼠的股骨中分离、培养和扩增BMSCs。H2O2刺激mBMSCs建立氧化应激损伤模型。实验分为对照组(不予H2O2处理)、不同浓度H2O2(100、200、300、400、500、800、1 000μmol/L处理mBMSCs)损伤组(n=5)。采用MTT法检测24、48、72 h各组的细胞活力;倒置显微镜观察BMSCs的形态学改变;采用细胞核Hoechst33342染色观察凋亡细胞核形态;Western blot检测各组Bcl-2、Bax、mTOR及其下游蛋白以及蛋白的磷酸化的表达。结果 100～1 000μmol/L浓度的H2O2作用mBMSCs24 h后,其形态学和病理学发生浓度依赖性的改变。H2O2浓度在100～300μmol/L时,随着H2O2浓度的增高,mBMSCs的mTOR、p70S6K、S6的表达水平有增高的趋势,磷酸化水平明显增高(P0.01),抗凋亡蛋白Bcl-2表达增高(P0.01),凋亡蛋白Bax表达不明显(P0.05)。H2O2浓度在400μmol/L以上时,随着H2O2浓度的增高,p-mTOR、p-p70S6K、p-S6、BCL-2的表达水平降低(P0.05,P0.01),而mTOR、p70S6K、S6蛋白变化不明显,同时Bax的表达水平明显增高(P0.01)。结论一定强度的氧化应激可以降低mBMSCs存活率,促进细胞凋亡,其机理可能与抑制mTOR及其下游信号通路的活性和抗凋亡蛋白Bcl-2表达,促进凋亡蛋白Bax表达有关。
[Abstract]:Objective To study the changes of mammalian target to frapamycin (mTOR) and its downstream signaling pathway in oxidative stress injury of mouse bone marrow stem cells (mBMSCs). The model of oxidative stress injury was established by stimulating mBMSCs. The experiment was divided into control group (without H2O2 treatment) and injury group (n=5) with different concentrations of H2O2 (100,200,300,400,500,800,1 000 micromol/L treatment). Cell viability was detected by MTT assay at 24,48,72 hours; morphological changes of BMSCs were observed by inverted microscope; and apoptosis was observed by Hoechst 33342 staining. The expression of Bcl-2, Bax, mTOR and its downstream proteins and protein phosphorylation were detected by Western blot. Results The morphology and pathology of mBMSCs changed in a concentration-dependent manner after 24 h exposure to H2O2 at 100-1 000 micromol/L concentration. The expression levels of 6K, S6, Bcl-2, Bcl-2 and Bax increased significantly (P 0.01). When the concentration of H2O2 was above 400 micromol/L, the expression levels of p-mTOR, p-p70S6K, p-S6 and BCL-2 decreased with the increase of H2O2 concentration (P 0.05, P 0.01). Conclusion A certain intensity of oxidative stress can reduce the survival rate of mBMSCs and promote cell apoptosis. The mechanism may be related to inhibiting the activity of mTOR and its downstream signaling pathway and the expression of anti-apoptotic protein Bcl-2 and promoting the expression of apoptotic protein Bax.
【作者单位】：重庆医科大学神经科学研究中心;第三军医大学大坪医院野战外科研究所第一研究室创伤、烧伤与复合伤国家重点实验室;第三军医大学大坪医院野战外科研究所第四研究室创伤、烧伤与复合伤国家重点实验室;
【基金】：国家重点基础研究发展计划(973计划,2012CB518104) 国家重点实验室基金(SKLZZ200907) 重庆市科委回国人员启动基金(CSTC2010BB5036)~~
【分类号】：R329.25

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