目的 观察骨髓间充质干细胞(MSCs)自噬活性的改变对其向成骨细胞分化能力的影响,初步探讨自噬在骨髓MSCs成骨分化中的作用.方法 不同浓度的雷帕霉素处理骨髓MSCs 48 h后,向成骨细胞诱导分化2周.免疫荧光激光共聚焦法检测LC3表达;单丹磺酰戊二胺(MDC)荧光染色检测自噬囊泡;Western blot检测LC3-Ⅰ和LC3-Ⅱ的表达;实时定量PCR检测成骨相关基因ALP和Runx2表达;Von Kossa染色检测钙质沉积程度改变.结果 经雷帕霉素处理后,激光共聚焦和Western blot检测均可见骨髓MSCs中LC3的表达明显增加;自噬囊泡数量增加;成骨相关基因ALP和Runx2的mRNA表达量明显减少;Von kossa染色可见MSCs钙质沉积程度减弱.上述改变呈剂量依赖性.结论 骨髓MSCs的基础自噬活性较低,雷帕霉素能够增加骨髓MSCs的自噬活性,但同时减弱其向成骨细胞分化的潜能.自噬可能参与骨髓MSCs向成骨细胞分化的过程.%Objective To observe the change of osteogenic potential of bone marrow derived mesenchymal stem cells (MSCs)after pretreatment with rapamycin which activates the autophagic activity,so as to explore the role of autophagy in the osteogenic differentiation of MSCs. Methods MSCs were induced to osteogenic differentiation for two weeks after pretreatment with rapamycin for 48 h. Immunocytochemistry with confocal technique was used to assess the localization of LC3. Autophagic vacuoles were characterized by monodansylcadaverine(MDC)staining. Real time RT PCR was employed to detect the expression of alkaline phosphatase(ALP) and Runx2. The expression levels of LC3 I and LC3 Ⅱ were detected by using Western blot. Calcium deposit was assessed by Von Kossa staining. Results After treatment with rapamycin,the expression of LC 3 was sig nificantly increased in MSCs. The number of autophagic vacuoles in MSCs was also increased. The mRNA expression levels of osteogenesis related genes ALP and Runx2 were decreased,and calcium deposit was reduced after pretreatment with rapamycin. These changes were dose dependent. Conclusion The bone marrow deprived MSCs possess a relatively low basic level of auto phagy. Rapamycin promoted autophagic activity of MSCs,while weakened the osteogenic differentiation potential of MSCs. Au tophagy may participate in the osteogenic differentiation of MSCs.
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