自噬对小鼠骨髓间充质干细胞干性标记基因的影响

摘要

AIM:To study the effect of autophagy on the expression of stemness marker gene Nanog,Oct-4 and Sox-Ⅱ in mouse bone marrow mesenchymal stem cells(BMSCs).METHODS:Mouse BMMSCs were isolated, cultured and identified by flow cytometry analysis.Autophagy of BMSCs was induced by rapamycin or inhibited by siR-NA targeting beclin-1 (si-beclin 1 ),the level of autophagy of BMMSCs and the expression of Nanog,Oct-4 and Sox-Ⅱ were examined by Real Time PCR (RT-PCR).RESULTS:The cultured BMMSCs expressed CD90.2 (97.4%),CD29 (97.6%),SCA-1 (83.5%),CD31(2.9%),CD146 (2.4%)and CD11b (5.6%).Treatment with rapamycin increased Nanog,Oct-4 and Sox-Ⅱexpression (P<0.05),whereas,si-beclin 1 decreased the auto-phagy acticity and down-regulated Nanog,Oct-4 and Sox-Ⅱ expression (P<0.05)of BMMSCs.CONCLUSION:Increasing autophagy activity of BMMSCs may be favourable for the cells to maintain the stemness.%目的：研究细胞自噬水平改变后，对小鼠骨髓间充质干细胞（bone marrow mesenchymal stem cells，BMMSCs）干性标记基因Nanog、Oct-4、Sox-Ⅱ表达的影响。方法：分离培养小鼠BMMSCs，经表型鉴定后，分别利用自噬激活剂雷帕霉素（rapamycin ）和靶向作用于 beclin 1的 si-RNA （small interfernce RNA target beclin 1，si-beclin 1）激活或抑制小鼠 BMMSCs 的自噬活性；利用实时定量 PCR 仪（Real-time PCR， RT-PCR）检测各组BMMSCs的各自噬相关基因以及干性标记基因的表达水平。结果：流式细胞仪检测结果显示，小鼠BMMCs中 CD90．2、CD29、SCA-1的阳性率分别为97．4％、97．6％、83．5％，CD31、CD146、CD11b的阳性率分别为2．9％、2．4％、5．6％；雷帕霉素激活BMMSCs自噬后，其干性标记基因Nanog、Oct-4、Sox-Ⅱ的表达均较对照组明显升高（P＜0．05），而用si-beclin 1抑制BMMSCs的自噬活性后，Nanog、Oct-4、Sox-Ⅱ的表达均较对照组明显下降（P＜0．05）。结论：提高BMMSCs的自噬水平有助于保持干细胞的特异性基因表达。