【摘要】：目的:探討骨髓間充質(zhì)干細(xì)胞(BMSCs)向神經(jīng)細(xì)胞分化前后自噬水平的變化,進(jìn)一步研究BMSCs神經(jīng)分化機(jī)制。方法:原代培養(yǎng)大鼠BMSCs,三代后用L-DMEM(2%DMSO+200μmol/L BHA)誘導(dǎo)其向神經(jīng)細(xì)胞分化,采用免疫細(xì)胞化學(xué)方法檢測(cè)神經(jīng)元特異標(biāo)記物NSE表達(dá),采用RT-PCR方法檢測(cè)自噬相關(guān)基因LC3B、Beclin1、Atg5、Atg7、Atg10的表達(dá),用Western blot及流式細(xì)胞術(shù)檢測(cè)了自噬標(biāo)志蛋白LC3B的表達(dá)。結(jié)果:大鼠BMSCs傳3代后,貼壁的BMSCs可達(dá)90%以上,類(lèi)成纖維細(xì)胞樣梭形生長(zhǎng),經(jīng)誘導(dǎo)劑作用5 h后,細(xì)胞呈現(xiàn)神經(jīng)細(xì)胞樣改變,NSE陽(yáng)性率為(83±5)%。RT-PCR結(jié)果表明BMSCs向神經(jīng)細(xì)胞分化后LC3B,Beclin1,Atg5,Atg7,Atg10的表達(dá)升高,流式細(xì)胞術(shù)檢測(cè)LC3B的平均熒光強(qiáng)度升高,Western blot結(jié)果表明神經(jīng)分化后LC3B-Ⅱ表達(dá)增加。結(jié)論:大鼠BMSCs向神經(jīng)細(xì)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(hào)：2332994