当归红芪超滤膜提取物诱导小鼠骨髓间充质干细胞分化为神经样细胞的实验研究

Ultrafiltration Membrane Extract Mixture from Angelica sinensis and Hedysarum polybotrys Induced Transdifferentiation of BMSCs in Mice: an Experimental Research

目的观察并评价当归红芪超滤膜提取物(简称中药合剂)诱导小鼠骨髓间充质干细胞(bone marrow derived stroma cells,BMSCs)分化为神经样细胞的效果。方法体外培养小鼠BMSCs后加入药物并分为5组:空白组、6g/L中药合剂组(简称低剂量组)、12g/L中药合剂组(简称高剂量组)及3g/L中药合剂联合0.5mmol/Lβ-巯基乙醇用药组(简称联合组)、β-巯基乙醇阳性对照组(简称对照组)。通过甲苯胺蓝染色观察各组诱导分化为神经样细胞的效果,应用免疫组织化学及免疫荧光技术检测分化后细胞所表达的神经元特异性烯醇化酶(neuron-specific enolase,NSE)、巢蛋白(nestin)、神经丝蛋白(neurofilament protein,NFP)、微管结合蛋白2(microtubule-associated protein-2,MAP2)及神经胶质原纤维酸性蛋白(glial fibrillary acidic protein,GFAP)5种神经特异性蛋白的差异,并应用流式细胞分析技术(flow cytometry,FCM)检测各组细胞生长周期的变化。结果诱导后细胞形态发生神经样细胞改变,两个中药合剂组的形态特征性均弱于联合组和对照组;除空白组外,上述5种蛋白在各组的表达为阳性,表达程度均为对照组最高(P<0.05),联合组次之(P<0.05);经流式细胞术检测细胞增殖率比较:对照组最低(P<0.05),高、低剂量组较高(P<0.05),联合组次之(P<0.05)。结论中药合剂可较为有效地诱导小鼠BMSCs分化为神经样细胞,诱导能力虽弱于对照组,但对于分化后细胞的增殖作用明显。联合组可有效诱导BMSCs分化为神经样细胞并使细胞有较高的增殖能力,可能是用于临床研究目前较为理想的用药途径。

Objective To observe and evaluate the effect of transdifferentiation of bone marrow derived stroma cells （BMSCs） into nerve cells by ultrafiltration membrane extract mixture from Angelica sinensis and Hedysarum polybotrys . Methods The BMSCs in vitro cultured after treated by ultrafiltration membrane extract mixture from Angelica sinensis and Hedysarum polybotrys were divided into 5 groups, i.e., the blank group, the low dose group （6 g/L mixture）, the high dose group （12 g/L mixture）, the combination group （3 g/L mixture＋0.5 mmol/Lβ-mercaptoethanol）, and the positive control group （β-mercaptoethanol）. The effects of transdifferentiation of nerve cells were observed using toluidine blue staining in each group. The differences of 5 specific neuroproteins, i.e. neuron-specific enolase （NSE）, nestin, neurofilament protein （NFP）, microtubule associated protein 2 （MAP2）, and glial fibrillary acidic protein （GFAP） were detected using immunohistochemical technique and immunofluorescent technique respectively. The changes of the cell cycle were detected using flow cytometry （FCM）. Results After induction BMSCs changed morphologically. The morphological features were weaker in the high and low dose groups than in the combination group and the positive group. Except the blank group, the aforesaid 5 proteins expressed positively in the rest groups. Their expression levels were highest in the positive control group （P 0.05）, followed by the combination group （P 0.05）. As for the cell proliferation rate detected by FCM, it was the lowest in the positive control group, followed by high dose group, low dose group, and then the combination group （all P 0.05）. Conclusions The ultrafiltration membrane extract mixture from Angelica sinensis and Hedysarum polybotrys could effectively induce the transdifferentiation of BMSCs into nerve cells. Its inducing capacities were weaker in the positive control group, but it showed marked proliferation effects on differentiated cells. Therefore, the mixture might be a more ideal medication pathway for effectively inducing BMSCs′ transdifferentiation into nerve cells, which might have higher proliferation and be used for clinical research.