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(P<0.01),抗凋亡蛋白Bcl-2表达增高(P<0.01),凋亡蛋白Bax表达不明显(P>0.05)。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(P<0.05,P<0.01),而mTOR、p70S6K、S6蛋白变化不明显,同时Bax的表达水平明显增高(P<0.01)。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。
Objective To investigate the possible effects and changes of mammalian target of rapamy- cin （mTOR） and its downstream signaling pathway in mouse bone marrow stem cells （mBMSCs） induced by oxidative stress. Methods mBMSCs were isolated from bone marrows from 30 healthy Kunming adult male mice, cultured and expanded. An oxidative stress model of mBMSCs was established by different concentrations of H202 （100, 200, 300, 400, 500, 800 and 1 000 μmol/L）. Cell viability was detected by MTT assay, and morphological changes of BMSCs were observed by inverted microscopy. The nucleus apotosis were accessed by Hoechst 33342 staining. Western blotting was employed to evaluate the expression of Bcl-2, Bax, mTOR, p70S6K and S6, as well as phosphorylated roTOR, p70S6K and S6. Results The mBMSCs had pathophysiologic changes after 100 to 1 000 μmol/L H2O2treatment in a dose-dependent manner. When H2O2 was given at concentrations of 100 to 300 μmol/L, the protein expression of mTOR, p70S6K and S6 in mBMSCs tended to be increased in a dose-dependent fashion, while the expression of their phosphorylated forms and anti-apoptosis protein Bcl-2 were significantly increased （P 〈 0. 01 ）. But, the expression of apoptosis protein Bax was not obviously changed （P 〉 0. 05 ）. However, when H2 02 was given at concentrations over 400 μmoL/L, the expression of Bcl-2, p-roTOR, p-p70S6K and p-S6 proteins were in a dose-dependent decrease in mBMSCs （P 〈0. 05, P 〈 0. 01 ）, while the expression of mTOR, p70S6K and S6 protein was not visibly altered,whereas the expression of was obviously increased （P 〈 0. 01 ）. Conclusion Oxidative stress to some extent causes reduced survival and increased apoptosis in BMSCs. The underlying mechanisms may be partly due to suppression of mTOR and its downstream signaling, decreased expression of Bcl-2, and enhanced expression of Bax.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2013年第2期114-118,共5页
Journal of Third Military Medical University
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)~~
