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目的探讨地黄多糖对过表达Notch1(NICD)大鼠骨髓间充质干细胞(BMSCs)诱导分化及增殖的影响。方法以P3~P5代大鼠BMSCs为研究对象,将细胞随机分为正常对照组、空载体组和转染组。脂质体法转染Notch1(NICD)过表达真核载体,转染后以地黄多糖(0.2 mg/m L)进行诱导分化,转染和诱导后行细胞一般状态观察,采用免疫细胞化学法和Western blotting法检测Notch1(NICD)蛋白、神经元特异性烯醇化酶(NSE)和胶质纤维酸性蛋白(GFAP)表达,CCK-8检测细胞存活率。结果转染后转染组细胞似神经胶质样细胞形态改变,诱导后各组细胞似神经元样细胞形态结构,转染组显著。转染组Notch1和GFAP蛋白表达阳性率显著高于正常对照组和空载体组(P<0.05),而NSE在3组间差异无统计学意义(P>0.05);诱导后转染组NSE阳性率高于正常对照组和空载体组(P<0.05)。转染后与诱导后比较,转染组Notch1表达阳性率降低(P<0.05),且正常对照组与空载体组亦降低(P<0.05);转染组NSE表达阳性率明显升高(P<0.01),且正常对照组与空载体组明显升高(P<0.01);转染组GFAP表达阳性率明显降低(P<0.01)。CCK-8检测显示,地黄多糖诱导后,随时间延长各组细胞存活率略呈下降趋势。结论转染高表达Notch1(NICD)大鼠BMSCs具有向神经胶质样细胞分化倾向,地黄多糖显著抑制Notch1蛋白的表达,诱导BMSCs向神经元样细胞分化。
Objective To investigate the effects of Rehmannia glutinosa polysaccharide on differentiation and proliferation of bone marrow-derived mesenchymal stem cells (BMSCs) in overexpressed Notch1 (NICD) rats. Methods The P3 ~ P5 generation of rat BMSCs were randomly divided into normal control group, empty vector group and transfection group. Liposomes were transfected into Notch1 (NICD) overexpression eukaryotic vector and transfected into Rehmannia glutinosa polysaccharide (0.2 mg / mL) for differentiation. After transfected and induced, the general status of cells was observed. Immunocytochemistry and immunocytochemistry The expression of Notch1 (NICD) protein, neuron specific enolase (NSE) and glial fibrillary acidic protein (GFAP) were detected by Western blotting. Cell viability was detected by CCK-8. Results After transfection, the morphological changes of glial-like cells were observed in transfected cells. After induced, the morphology and structure of neuron-like cells in each group showed significant changes. The positive rates of Notch1 and GFAP in transfection group were significantly higher than those in normal control group and empty vector group (P <0.05), while there was no significant difference in NSE between the three groups (P> 0.05) Rate was higher than the normal control group and empty vector group (P <0.05). Compared with the control group, the positive rate of Notch1 expression in transfected group was lower (P <0.05), and the expression of Notch1 in transfected group was also lower (P <0.05). The positive rate of NSE in transfected group was significantly higher than that in control group <0.01). The positive rate of GFAP in transfected group was significantly lower than that in control group and blank vector group (P <0.01). CCK-8 test showed that after the induction of Rehmannia glutinosa, cell survival rate of each group decreased slightly with time. CONCLUSION: BMSCs transfected with high expression Notch1 (NICD) rat tend to differentiate into glial-like cells. Rehmannia polysaccharide significantly inhibits Notch1 protein expression and induces BMSCs to differentiate into neuron-like cells.