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目的探讨传统中药红景天苷诱导大鼠BMSCs向胆碱能神经细胞分化的作用及影响,以期为红景天苷应用于干细胞治疗神经系统疾病提供理论依据。方法取4～6周龄Wistar大鼠(体重约120 g)2只分离、培养BMSCs,并采用流式细胞仪鉴定。取第2代细胞,根据诱导方法不同将实验分为红景天苷诱导组(A组)、维甲酸诱导组(B组)和空白对照组(C组),A、B组BMSCs分别采用红景天苷(20μtg/mL)和维甲酸(5μmol/mL)诱导培养1、3、6、9 d,MTT法检测细胞增殖活力;细胞免疫荧光染色检测神经细胞相关标志分子神经元特异性烯醇化酶(neuron-specificenolase,NSE)、神经微管相关蛋白2(microtubule-associated protein 2,MAP2)、β-微管蛋白Ⅲ(β-TubulinⅢ)、神经胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)、乙酰胆碱(Acetylcholine,Ach)及NGF的表达;RT-PCR检测NSE、β-TubulinⅢ、GFAP、γ-氨基丁酸(γ-aminobutyric acid,GABA)、脑源性神经生长因子(brain derived neurotrophic factor,BDNF)mRNA的表达;ELISA法测定培养液中BDNF和NGF含量;Western blot检测NGF蛋白表达水平。结果流式细胞仪检测显示,CD90和CD106阳性,CD34和CD45阴性,实验细胞为BMSCs。A、B组诱导培养6 d和9 d,细胞增殖活力较C组明显增强(P<0.05)。RT-PCR检测示,A组诱导培养6 d,NSE、BDNF、β-TubulinⅢ、GFAP mRNA表达丰度上调至峰值。B组诱导培养6 d,NSE mRNA表达丰度上调至峰值;1 d时BDNF mRNA表达丰度上调,6 d时达峰值;3 d时β-TubulinⅢmRNA表达丰度上调至峰值;1 d时GFAP mRNA表达丰度达峰值,与其余各时间点及C组比较差异均有统计学意义(P<0.01)。各组各时间点均未见GABA mRNA表达。细胞免疫荧光染色示,诱导培养3 d,A、B组NSE、MAP2、β-TubulinⅢ和GFAP阳性率与C组比较差异均有统计学意义(P<0.01);A、B组诱导培养3、6、9 d,Ach阳性率均高于诱导培养1 d时及C组阳性率(P<0.01);A、B组各时间点NGF阳性率均显著高于C组(P<0.01)。ELISA法检测显示,A、B组诱导培养1、3、6、9 d时,BDNF、NGF表达水平均较C组上调(P<0.01),各时间点A、B组间BDNF表达水平比较差异无统计学意义(P<0.01)。Western blot检测显示,A组诱导培养6 d、B组诱导培养3 d时NGF蛋白表达最高。结论红景天苷能定向诱导大鼠BMSCs分化为胆碱能神经细胞。
Objective To investigate the effect of traditional Chinese medicine salidroside on differentiation of rat BMSCs into cholinergic neurons and to provide a theoretical basis for the application of salidroside in the treatment of nervous system diseases. METHODS: Two Wistar rats (weighing about 120 g), 4 to 6 weeks old, were isolated and cultured. BMSCs were cultured and identified by flow cytometry. The second passage cells were divided into three groups according to different induction methods: Salidroside inducing group (A group), Retinoic acid inducing group (B group) and blank control group (C group) The cells were cultured for 1, 3, 6, and 9 days with Sedumgenin (20μ tg / mL) and retinoic acid (5μmol / mL). Cell viability was detected by MTT assay. Neuron-specific enolase Neuron-specific enolase (NSE), microtubule-associated protein 2 (MAP2), β-tubulinⅢ, glial fibrillary acidic protein (GFAP) ), Acetylcholine (Ach) and NGF were detected by RT-PCR. The expressions of NSE, β-TubulinⅢ, GFAP, γ-aminobutyric acid (GABA) and brain derived neurotrophic factor , BDNF) mRNA were detected by enzyme-linked immunosorbent assay (ELISA). BDNF and NGF in culture medium were detected by ELISA. The expression of NGF protein was detected by Western blot. Results Flow cytometry showed that CD90 and CD106 were positive, CD34 and CD45 were negative, and experimental cells were BMSCs. The proliferation of cells in group A and B at 6 d and 9 d was significantly higher than that in group C (P <0.05). The expression of NSE, BDNF, β-TubulinⅢ and GFAP mRNA in group A was up-regulated to the peak by RT-PCR. In group B, the expression of NSE mRNA was up-regulated to the peak at 6 days after induction. The level of BDNF mRNA was up-regulated on day 1 and peaked at 6 days. The level of β-TubulinⅢ mRNA up-peaked at 3 days. The expression abundance peaked at the same time point, and the other time points and C group were significantly different (P <0.01). No GABA mRNA expression was found in each group at each time point. Cell immunofluorescence staining showed that the positive rates of NSE, MAP2, β-TubulinⅢ and GFAP in group A and group B were significantly different from those in group C (P <0.01) on day 3, The positive rate of Ach at 6 and 9 d was higher than that at 1 d and the positive rate of C (P <0.01). The positive rate of NGF in group A and B at each time point was significantly higher than that in group C (P <0.01). The results of ELISA showed that the expression of BDNF and NGF in group A and B were higher than that in group C at 1, 3, 6, and 9 d after induction (P <0.01). The expression of BDNF in group A and B at each time point was significantly different No statistical significance (P <0.01). Western blot results showed that the expression of NGF protein was the highest in group A when cultured for 6 days and that in group B for 3 days. Conclusion Salidroside can induce the differentiation of rat BMSCs into cholinergic cells.