【摘要】：目的:探討骨髓間充質(zhì)干細胞(BMSCs)向神經(jīng)細胞分化前后自噬水平的變化,進一步研究BMSCs神經(jīng)分化機制。方法:原代培養(yǎng)大鼠BMSCs,三代后用L-DMEM(2%DMSO+200μmol/L BHA)誘導其向神經(jīng)細胞分化,采用免疫細胞化學方法檢測神經(jīng)元特異標記物NSE表達,采用RT-PCR方法檢測自噬相關(guān)基因LC3B、Beclin1、Atg5、Atg7、Atg10的表達,用Western blot及流式細胞術(shù)檢測了自噬標志蛋白LC3B的表達。結(jié)果:大鼠BMSCs傳3代后,貼壁的BMSCs可達90%以上,類成纖維細胞樣梭形生長,經(jīng)誘導劑作用5 h后,細胞呈現(xiàn)神經(jīng)細胞樣改變,NSE陽性率為(83±5)%。RT-PCR結(jié)果表明BMSCs向神經(jīng)細胞分化后LC3B,Beclin1,Atg5,Atg7,Atg10的表達升高,流式細胞術(shù)檢測LC3B的平均熒光強度升高,Western blot結(jié)果表明神經(jīng)分化后LC3B-Ⅱ表達增加。結(jié)論:大鼠BMSCs向神經(jīng)細胞分化后自噬水平增加。
[Abstract]:Aim: to investigate the changes of autophagy level before and after (BMSCs) differentiation in bone marrow mesenchymal stem cells (BMSCs) and to further study the mechanism of BMSCs neural differentiation. Methods: rat BMSCs, was induced to differentiate into neural cells by L-DMEM (2%DMSO 200 渭 mol/L BHA) for three generations. The expression of neuron specific marker NSE was detected by immunocytochemistry. The expression of autophagy associated gene LC3B,Beclin1,Atg5,Atg7,Atg10 was detected by RT-PCR and the expression of autophagy marker protein LC3B was detected by Western blot and flow cytometry. Results: after 3 passages of rat BMSCs, more than 90% of BMSCs adherent to the wall. Fibroblast-like fusiform growth was observed. After 5 h of induction, the cells showed neuronal changes. The positive rate of NSE was (83 鹵5)%. The results of RT-PCR showed that the expression of LC3B,Beclin1,Atg5,Atg7,Atg10 increased after the differentiation of BMSCs into neurons, and the mean fluorescence intensity of LC3B was increased by flow cytometry. Western blot results showed that the expression of LC3B- 鈪，

本文編號：2332994