地黄多糖对大鼠骨髓间充质干细胞向神经样细胞诱导分化作用的研究

目的:探讨地黄多糖体外对大鼠骨髓间充质干细胞( BMSCs)分化为神经样细胞的诱导作用.方法:以大鼠BMSCs为研究对象,采用流式细胞仪检测BMSCs表面标志,MTT法检测细胞生长曲线,当第3代细胞爬片达到80％以上汇合时,分为对照组、化学方法诱导组(5 mmol·L -1,β-mercap toethanol)、脑源性神经生长因子(10 μg·L-1,BDNF)组和地黄多糖组(200 mg·L-1)进行诱导,6h后应用免疫细胞化学染色法检测各实验组神经元巢蛋白(nestin)、神经元特异性烯醇化酶(NSE)、胶质纤维酸性蛋白(GFAP)的表达,RT-PCR检测nestin,NSE,βⅢ-微管蛋白(βⅢ-tubulin)和GFAP mRNA的表达情况.结果:经分离鉴定培养5代以内大鼠BMSCs增殖旺盛、具有较好的细胞活力；免疫细胞化学染色和RT-PCR检测结果显示,对照组不表达神经细胞标志物,各诱导组诱导后有特异性神经细胞标志物表达.其中地黄多糖组Nestin,NSE阳性细胞率分别为( 56.74±1.36)％,(73.37±1.27)％,高于化学诱导组(28.21±2.43)％,(2.31±2.72)％和神经生长因子组(31.3±1.61)％,(28.87±1.65)％,(P＜0.05)；GFAP阳性细胞率(20.17±1.27)％低于化学诱导组和神经生长因子组,差异具有统计学意义(P＜0.05)；化学诱导组Nestin,NSE和GFAP阳性细胞率与生长因子组比较,差异无统计学意义.结论:地黄多糖具有诱导BMSCs向神经样细胞分化的作用,主要向神经元样细胞方向诱导.

Study on Differentiation of Rat Bone Marrow Mesenchymal Stem Cells into Neuron-like Cells Induced by Rehmannia glutinosa Polysaccharide in vitro

Objective:To investigate the effect of Rehmannia glutinosa polysaccharide(RGP) on inducing differentiation of rats bone marrow mesenchymal stem cells(BMSCs) into neuron-like cells in vitro.Method:After characterized cell surface markers by flow cytometry analysis and assessed cell growth curve by MTT,rat BMSCs were experimented as the object.When the third passage cells grew to fusion of more than 80%,the cells were divided into 4 groups such as the control group,the β-mercap toethanol(BME)induction group,the brain derived neurotrophic factor(BDNF) induction group and the(RGP) induction group.Immunocytochemical stain and RT-PCR method were used to detect several neural specific proteins such as nestin,neuronal specific endase(NSE),glial fibrillory acidic protein(GFAP) and the related mRNA genes expression of nestin,βⅢ-tubulin,NSE and GFAP respectively after 6 hours.Result:Rat BMSCs had good vitality which were isolated,identified and cultivated within five generations.The detection of immunocytochemical stain and RT-PCR method show that neural cell markers were not expressed in the control group,but expressed in the other groups.Positive cells rate of nestin and NSE in the RGP induction group(56.74±1.36)%,(73.37±1.27)% respectively was higher than the BME induction group(28.21±2.43)%,(2.31±2.72)%and the BDNF induction group(31.3±1.61)%,(28.87±1.65)%(P<0.05)and positive cells rate of GFAP(20.17±1.27)%lower(P<0.05),but there was no difference between the BME induction group and the BDNF induction group in nestin,NSE and GFAP positive cells rate.Conclusion:The rat bone marrow mesenchymal stem cells could be differentiated into neure-like cells in vitro with the induction of R.glutinosa polysaccharide,mainly inducted to neuron-like cell.