Zhang Ming, Zhao Hongbin, Li Zhiyun, Yang Yinshu, Wen Yimin, Dong Juzi, Zhang Quanwei, Ge Baofeng
Institute of Orthopedics, General Hospital of Lanzhou Military Command, Lanzhou Gansu, 730050, PR China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2012 Feb;26(2):158-65.
To investigate the effect of salidroside on rat bone marrow mesenchymal stem cells (BMSCs) differentiation into the cholinergic nerve cells, so as to provide the theory basis of the combination of salidroside and stem cells for clinical therapy of nervous system diseases.
BMSCs were isolated from 2 Wistar rats (aged 4-6 weeks,weighing 120 g), which were identified by CD34, CD45, CD90, and CD106 with flow cytometry. According to inducing method, BMSCs at passage 2 were divided into 3 groups: In groups A and B, BMSCs were induced by salidroside (20 microg/mL) and retinoic acid (5 micromol/mL) respectively for 1, 3, 6, and 9 days, in group C, BMSCs were cultured with serum-free DMEM/F12 medium as control. MTT assay was used to detect the cellular proliferation activity. The immunofluorescence chemical technology was used to detect the expressions of nerve growth factor (NGF) and relevant marker molecule of nerve cells, including neuron-specific enolase (NSE), microtubule-associated protein 2 (MAP2), beta-Tubulin III, glial fibrillary acidic protein (GFAP), and the marker of cholinergic neuron, such as Acetylcholine (Ach) and NGF. RT-PCR was used to detect mRNA expressions of NSE, beta-Tubulin III, GFAP,brain derived neurotrophic factor (BDNF),and gamma-aminobutyric acid (GABA). ELISA was used to detect the levels of BDNF and NGF, and the expression level of NGF protein was analyzed by Western blot.
The results of the flow cytometry showed that the cultured cells were CD90 and CD106 positive, and CD34 and CD45 negative,which indicated that the cells were BMSCs. The cellular proliferation activity in groups A and B were significantly higher than that in group C at 6 days and 9 days (P < 0.05). RT-PCR results showed that the expression level of NSE,BDNF, beta-Tubulin III,GFAPmRNA were increased in group A at 6 days; In group B, that expression level of NSE mRNA was up-regulated at 6 days, that expression level of BDNF mRNA increased at 1 days and reached the peak at 6 days, and that expression level of beta-Tubulin III mRNA was up-regulated at 3 days, which was significantly higher than that at the other time points, and than that in group C (P < 0.01). But no GABA mRNA expression was detected in each group. Immunofluorescence chemical technology staining showed that the positive rates of NSE, MAP2, beta-Tubulin III, and GFAP were significantly higher in group A than those in group C at 3 days; the positive rates of Ach were significantly higher at 3, 6, and 9 days than those at 1 day in groups A and B, and in groups A and B than in group C (P < 0.01); the positive rates of NGF in groups A and B were significantly higher than those in group C (P < 0.01). The levels of BDNF and NGF in groups A and B were significantly higher than those in group C at 1, 3, 6, and 9 days (P < 0.01), but no significant difference of BDNF was found between groups A and B (P > 0.05). The expression level of NGF protein in groups A and B were significantly higher than that in group C (P < 0.01). The NGF expression reached the peak at 6 days in group A and at 3 days in group B.
Salidroside could induce rat BMSCs differentiate into cholinergic nerve cells in vitro.
研究红景天苷对大鼠骨髓间充质干细胞（BMSCs）向胆碱能神经细胞分化的影响，为红景天苷与干细胞联合用于神经系统疾病的临床治疗提供理论依据。
从2只Wistar大鼠（4 - 6周龄，体重120 g）分离BMSCs，通过流式细胞术用CD34、CD45、CD90和CD106进行鉴定。根据诱导方法，将第2代BMSCs分为3组：A组和B组分别用红景天苷（20 μg/mL）和视黄酸（5 μmol/mL）诱导1、3、6和9天，C组用无血清DMEM/F12培养基培养作为对照。采用MTT法检测细胞增殖活性。用免疫荧光化学技术检测神经生长因子（NGF）和神经细胞相关标志物分子的表达，包括神经元特异性烯醇化酶（NSE）、微管相关蛋白2（MAP2）、β-微管蛋白III、胶质纤维酸性蛋白（GFAP）以及胆碱能神经元标志物，如乙酰胆碱（Ach）和NGF。用RT-PCR检测NSE、β-微管蛋白III、GFAP、脑源性神经营养因子（BDNF）和γ-氨基丁酸（GABA）的mRNA表达。用ELISA检测BDNF和NGF水平，并用Western blot分析NGF蛋白的表达水平。
流式细胞术结果显示，培养的细胞CD90和CD106阳性，CD34和CD45阴性，表明这些细胞为BMSCs。A组和B组在第6天和第9天的细胞增殖活性显著高于C组（P < 0.05）。RT-PCR结果显示，A组在第6天NSE、BDNF、β-微管蛋白III、GFAP mRNA表达水平升高；B组在第6天NSE mRNA表达水平上调，BDNF mRNA表达水平在第1天升高并在第6天达到峰值，β-微管蛋白III mRNA表达水平在第3天上调，显著高于其他时间点及C组（P < 0.01）。但各组均未检测到GABA mRNA表达。免疫荧光化学技术染色显示，A组在第3天NSE、MAP2、β-微管蛋白III和GFAP的阳性率显著高于C组；A组和B组在第3、6和9天Ach的阳性率显著高于第1天，且A组和B组高于C组（P < 0.01）；A组和B组NGF的阳性率显著高于C组（P < 0.01）。A组和B组在第1、3、6和9天BDNF和NGF水平显著高于C组（P < 0.01），但A组和B组之间BDNF无显著差异（P > 0.05）。A组和B组NGF蛋白表达水平显著高于C组（P < 0.01）。A组NGF表达在第6天达到峰值，B组在第3天达到峰值。
红景天苷可在体外诱导大鼠BMSCs分化为胆碱能神经细胞。
