体外诱导大鼠骨髓间充质干细胞分化为胆碱能样神经元  

Differentiation of rat marrow mesenchymal stem cells into cholinergic like-neurons by inducing reagents

作　　者：井绪东[1] 张洹[1] 陈丽[1] 何冬梅[1] 

机构地区：[1]暨南大学医学院血液病研究所,广东广州510632

出　　处：《中国老年学杂志》2010年第19期2775-2778,共4页Chinese Journal of Gerontology

基　　金：国务院侨办重点学科建设基金资助(51205002)

摘　　要：目的探讨大鼠骨髓间充质干细胞能否分化为胆碱能样神经元。方法分离纯化大鼠骨髓间充质干细胞(MSCs),取2～4代MSCs,进行诱导其向胆碱能神经元分化。诱导方案:10%胎牛血清诱导2d;换液为DMEM/F12培养基,加入碱性成纤维细胞生长因子(bFGF)、巯基乙醇(ME)、维甲酸(RA)、神经生长因子(NGF),诱导8d;加入表皮生长因子(EGF),肝素(Heparin)继续诱导8d。空白对照组不作任何处理。观察诱导后的形态变化。采用RT-PCR检测巢蛋白(nestin),核受体相关因子(Nurr1),胆碱乙酰转移酶(ChAT)mRNA的表达,间接免疫荧光法检测表达nestin、ChAT、神经核蛋白(NeuN),乙酰胆碱酯酶(AchE)的阳性细胞。结果诱导后细胞形态逐渐由长梭形变为圆形或椭圆形,突起形成;RT-PCR显示,诱导后的细胞高表达nestin、Nurr1、ChAT,且基因表达相对强度与未诱导的MSCs比较有显著差异(P<0.01)。免疫荧光法检测显示,诱导后的细胞高表达nestin、ChAT、NeuN、AchE,且阳性细胞大于80%,而对照组除ChAT阳性细胞为1%外,其他均为阴性。结论大鼠MSCs可诱导为胆碱能样神经细胞。Objective To explore the differentiation of rat marrow mesenchymal stem cells （MSCs） into cholinergic like-neurons by inducing reagents. Methods MSCs from rat bone marrow were isolated and purified by cell culture,which were induced and differentiated into cholinergic like-neurons with 2~4 generations of MSCs by inducing reagents. The protocol was as follows：MSCs were incubated with 10% FBS for 2 days and then medium was replaced by DMEM/F12 complemented with 10 μg/L of bFGF,0.1 mmol/L of 2-ME,1 mmol/L of RA,10 μg/L of NGF for 8 days. And then 10 μg/L of EGF and 1 250 U/ml of heparin were added into the culture system for culture for 8 days. RT-PCR and gel electrophoresis were used to detect gene expression of nestin,Nurr-1,choline acetyltransferase （CHAT）. Immunological fluorescent staining was used to measure nestin,CHAT,NeuN,acetylcholine esterase （AchE） positive cells. Results Induced cells gradually became from long shuttle into round or ellipse with neuron-like dendrite. RT-PCR showed that the induced neuron-like cells expressed special neurocyte genes of nestin,Nurron,CHAT. Indirect immunological fluorescent staining showed positive cells with nestin,CHAT,NeuN,AchE reached 80% respectively. Conclusions MSCs from rat bone marrow can differentiate into cholinergic like-neurons.

关 键 词：间充质干细胞 胆碱能神经元 大鼠 生长因子 

分 类 号：R734[医药卫生—肿瘤]