中药诱导大鼠骨髓间质干细胞分化的实验

背景体外定向诱导分化可使骨髓间质干细胞(mesenchymal stem cells,MSCs)转化为成骨细胞、脂肪细胞、软骨、肌肉细胞、神经元样细胞等,而中药是否能对大鼠骨髓间质干细胞有诱导分化作用呢?目的研究SD大鼠MSCs体外扩增培养及用中药定向诱导分化为神经元样细胞的功效.设计以细胞为研究对象,重复观察测量,探索性研究.单位一所医学院病理生理学教研室.材料实验用骨髓取自经检验健康的SD雄性大鼠.方法通过贴壁法分离大鼠MSCs,体外扩增培养,流式细胞仪检测其表面抗原表达,多种中药成分定向诱导MSCs分化为神经元样细胞.光镜下观察细胞形态,免疫细胞化学法检测神经细胞特异性抗原标志.主要观察指标①MSCs分离和扩增结果.②MSCs表面抗原及神经元样细胞鉴定结果.结果大鼠MSCs可通过贴壁法成功分离并可在体外大量扩增.流式细胞仪检测结果显示CD14,D11α,D34,D38,D45,D80,D86为阴性,D29,D44,D90,D105,D166呈阳性.黄芪、天麻、人参、当归、脑新舒、人参蜂王浆等多种中药诱导1～3 h后大部分MSCs转变为神经元样细胞,出现胞体和突起,免疫细胞化学染色神经元特异性烯醇酶、巢蛋白呈阳性,胶质纤维酸性蛋白阴性.结论SD大鼠骨髓间质干细胞是具多向分化潜能的细胞,具有较强的增殖、自我更新能力.在适宜的诱导条件下,可以分化为神经元样细胞.

Chinese herbs for inducing differentiation of mesenchymal stem cells in rats

BACKGROUND: By targeting inducing differentiation in vitro,mesenchymal.stem cells(MSCs) transform into osteoblasts,lipocytes,chondrocytes,muscular cells,neuronal cells,etc. Whether Chinese herbs act on induced differentiation of MSCs in rats or not?OBJECTIVE: To study the amplification of MSCs cultured in vitro in SD rats and efficacy of Chinese herbs on targeting inducing differentiation of neuron-like cells.DESIGN: Exploring study with repeated observation and measurement based on cells.SETTING: Department of pathophysiology in a medical college.MATERIALS: Experimental marrow collected from SD male tested-healthy rats.METHODS: By adhesion method,MSCs in rats were isolated for amplifying culture in vitro. Flow-type cell instrument was applied for the determination of its surface antigen expression. Various Chinese herbal components were used for the targeting inducing differentiation of MSCs into neuron-like cells. The cellular morphology was observed under optical microscope. and the specific antigen label of neuronal cells was determined with immunocyto-chemical method.MAIN OUTCOME MEASURES:①Results of MSCs isolation and amplification;②Results of identification of MSCs surface antigen and neuon-like cells.RESULTS: By adhesion method,MSCs in rats were isolated successfully and amplified in a large amount in vitro. It was indicated in the results determined by flow-type cell instrument that CD14,CD1 1α,CD34,CD38,CD45,CD80 and CD86 presented negative,and CD29,CD44,CD90,CD105 and CD166 presented positive. By induced with various kinds of Chinese herbs,like huangqi(Radix Astragali seu Hedysari),tianma(Rhizoma Gastrodiae),renshen (Radix Ginseng),danggui(Radix Angelicae Sinensis),naoxinshu,renshen fengwangjian for 1 to 3 hours,most MSCs transformed into neuron-like cells,presenting soma and neurite. With immunocyto-chemical staining,neuron-specific enolase(NSE) and nestin displayed positive and glial fibrillary acidic protein negative.CONCLUSION: MSCs in SD rats have the potential of multi-targeting differentiation,presenting a strong capacity of amplification and self-replacement. In a suitable inducing condition,MSCs may differentiate into neuron-like cells.