左归丸及其拆方对大鼠骨髓间充质干细胞向神经样细胞分化的影响

Effects of Zuogui Pills and its decomposed recipes on the differentiation of bone marrow mesenchymal stem cells into neuron-like cells

目的:观察左归丸及其拆方对大鼠骨髓间充质干细胞(BMSCs)向神经样细胞分化的影响。方法:采用全骨髓贴壁培养法培养、扩增BMSCs;观察细胞形态,采用流式细胞术检测细胞表面CD90、CD29、CD34和CD45表达以鉴定BMSCs;分别使用β-巯基乙醇、左归丸、左归丸8个拆方药物血清分别诱导BMSCs分化,采用免疫细胞化学法观察诱导后细胞表面神经标志蛋白Nestin、NSE和GFAP表达;Real-time PCR法检测诱导后干细胞因子(SCF)mRNA表达。结果:培养至第3代的BMSCs表面CD90和CD29表达呈阳性,CD34和CD45表达呈阴性;经β-巯基乙醇、左归丸及其拆方药物血清分别诱导后,BMSCs可见典型的神经元样改变,与空白对照组比较,各诱导组神经标志蛋白Nestin、NSE和GFAP表达均显著增高(P<0.05),SCF mRNA表达有升高的趋势。结论:左归丸及其拆方可诱导大鼠BMSCs分化,分化细胞具备神经细胞的形态及特征,且诱导后部分干细胞因子表达明显升高。

Objective： To observe the effects of Zuogui Pills and its decomposed recipes on the differentiation of rat bone marrow mesenchymal stem cells （BMSCs） into neuron-like cells. Methods： BMSCs were cultured and expanded by whole bone marrow adherent culture method. Cell morphology was observed. The expression of CD90, CD29, CD34 and CD45 on cell surface was detected by flow cytometry to identify BMSCs. 13 -mercaptoethanol, Zuogui Pills serum and Zuogui Pills decomposed recipes （a total of 8 prescriptions） serum were used respectively to induce the differentiation of BMSCs. The expressions of Nestin, NSE and GFAP were detected by immunocytochemistry. The stem cell factor （SCF） mRNA expression of stem cells after induction was detected by Real-time PCR. Results： The expression of CD90 and CD29 on the surface of BMSCs cultured to the third generation was positive, and the expression of CD34 and CD45 was negative. After induction by 13 -mercaptoethanol, Zuogui Pills and its decomposed recipes, BMSCs showed typical neuron-like changes. Compared with the blank control group, the expression of Nestin, NSE and GFAP were significantly increased in each induction group （P〈0.05）, and the expression of SCF mRNA was also increased, but there was no significant difference. Conclusion： Zuogui Pills and its decomposed formula can induce the differentiation of rat BMSCs. The differentiated cells have the morphology and characteristics of nerve ceils, and the expression of stem cell factor is significantly increased after induction.