【摘要】 目的:探讨骨髓间充质干细胞(BMSCs)向神经细胞分化前后自噬水平的变化,进一步研究BMSCs神经分化机制。方法:原代培养大鼠BMSCs,三代后用L-DMEM(2%DMSO+200μmol/L BHA)诱导其向神经细胞分化,采用免疫细胞化学方法检测神经元特异标记物NSE表达,采用RT-PCR方法检测自噬相关基因LC3B、Beclin1、Atg5、Atg7、Atg10的表达,用Western blot及流式细胞术检测了自噬标志蛋白LC3B的表达。结果:大鼠BMSCs传3代后,贴壁的BMSCs可达90%以上,类成纤维细胞样梭形生长,经诱导剂作用5 h后,细胞呈现神经细胞样改变,NSE阳性率为(83±5)%。RT-PCR结果表明BMSCs向神经细胞分化后LC3B,Beclin1,Atg5,Atg7,Atg10的表达升高,流式细胞术检测LC3B的平均荧光强度升高,Western blot结果表明神经分化后LC3B-Ⅱ表达增加。结论:大鼠BMSCs向神经细胞分化后自噬水平增加。更多还原

【Abstract】 Objective: To study the autophagy activity between rat bone marrow stem cells( BMSCs) neural differentiation in order to explore the mechanism involve in this process. Methods: BMSCs were passed by 3 generation,then was induced with the revulsant 2%( DMSO) + 200 μmol / L( BHA),NSE expression was detected by immunocytochemical stain,the mRNA expression of autophagy associated genes LC3 B,Beclin1,Atg5,Atg7,Atg10 were detected by RT-PCR,the autophagy protein LC3 B was examined by Western blot and flow cytometry analysis. Results: BMSCs were passed by 3 generation,the purity of BMSCs could reach more than90%,the morphology of cells were like fibroblasts,after the revulsant 2% DMSO + 200 μmol / L BHA induced,cells were extended long neurites,like nerve cells,positive rate of NSE staining was( 83 ± 5) %,RT-PCR results showed that the expression of autophagy associated genes LC3 B,Beclin1,Atg5,Atg7 Atg10 were rised after BMSCs neural differentiation,Western blot analysis showed that the LC3B-Ⅱ protein expression was increased after neural differentiation and the MFI of LC3 B was highten by flow cytometry. Conclusion: Autophagy is increased after rat BMSC neural differentiation.更多还原