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背景:既往研究证明益气活血方补阳还五汤具有抑制脑缺血及再灌注模型细胞凋亡、抗自由基等作用,但关于本方剂对干细胞分化影响的报道较少。目的:观察益气活血方体外诱导大鼠骨髓间充质干细胞向神经细胞分化的作用。设计、时间及地点:以细胞为对象,对比观察实验,于2006-03/2008-02在河南省中医药研究院国家中医管理局三级实验室进行。材料:清洁级一二月龄SD大鼠20只,雌雄各半,体质量100～150g。益气活血方由本院制剂室生产,选用当归60g,桃仁30g,川芎30g加水720mL,提取挥发油,备用。药渣及提取液加入黄芪1200g,赤芍60g,地龙30g,红花30g,再加入920mL水,煎煮1h,滤过,药渣再加入8640mL水,煎煮1h,滤过,合并2次煎液,浓缩至600mL,加入挥发油及吐温-803mL,混匀,装瓶进行高压高温消毒。每毫升相当于生药2.4g。方法:采用密度梯度离心法分离获取SD大鼠骨髓单个核细胞层,将细胞经过传代培养使骨髓间充质干细胞得到扩增和纯化。将第10代的细胞悬液接种于直径40mm塑料培养皿中,细胞生长达80%～90%融合时,加入含体积分数为0.10的FBS、碱性成纤维细胞生长因子的DMEM培养基培养24h,益气活血方组而后换为含诱导液二甲亚砜、丁羟茴醚、β-巯基乙醇、益气活血方诱导5h,对照组换为含诱导液二甲亚砜、丁羟茴醚、β-巯基乙醇诱导5h。主要观察指标:采用流式细胞仪鉴定骨髓间充质干细胞;用免疫细胞化学方法检测诱导后细胞巢蛋白、神经元特异性烯醇化酶、胶质纤维酸性蛋白的表达。结果:经流式细胞仪检测显示,细胞表面标志CD90、CD106阳性,CD45、CD34呈阴性。免疫细胞化学方法检测显示,益气活血方组在诱导1,3h分化巢蛋白细胞与诱导5h分化的神经元特异性烯醇化酶细胞和胶质纤维酸性蛋白细胞,与对照组相比,差异显著(P<0.01)。结论:益气活血方具有体外定向诱导骨髓间充质干细胞向神经细胞分化的作用。
BACKGROUND: Previous studies have demonstrated that BYQHQF has the effects of inhibiting apoptosis and anti-free radicals in cerebral ischemia and reperfusion models, but there are few reports on the effects of this prescription on stem cell differentiation. Objective: To observe the effect of Yiqi Huoxue decoction on the differentiation of rat bone marrow mesenchymal stem cells into neural cells in vitro. DESIGN, TIME AND SETTING: The cells were used as subjects and contrast observation experiments were performed at the National Laboratory for Traditional Chinese Medicine, Henan Provincial Institute of Traditional Chinese Medicine, from March 2006 to February 2008. MATERIALS: Twenty male SD rats of clean grade one-two-month-old were divided into males and females with a body weight of 100-150 g. Yiqi Huoxue Fang is produced by the preparation room of this hospital. Angelica 60g, peach kernel 30g, and Chuanxiong 30g water 720mL are used to extract volatile oil. Dregs and extracts were added Astragalus 1200g, red peony 60g, ground dragon 30g, safflower 30g, then add 920mL water, boiling 1h, filtration, dregs and then add 8640mL water, boiling 1h, filtration, combined 2 times Decoction, concentrated to 600mL, adding volatile oil and Tween-803mL, mixing, bottling high-pressure high temperature sterilization. Each milliliter is equivalent to crude drug 2.4g. METHODS: SD rat bone marrow mononuclear cell layer was isolated by density gradient centrifugation, and the cells were subcultured to amplify and purify bone marrow mesenchymal stem cells. The 10th generation cell suspension was inoculated into a 40 mm diameter plastic petri dish. When the cell growth was 80% to 90% confluent, the cells were incubated with DMEM containing 0.1% FBS and basic fibroblast growth factor for 24 hours. , Yiqi Huoxue Decoction group was changed to induction solution containing dimethyl sulfoxide, butylated hydroxyanisole, β-mercaptoethanol, Yiqi Huoxue Fang induced 5h, the control group was replaced with inducer dimethyl sulfoxide, butylated hydroxy anisole , β-mercaptoethanol induced 5h. MAIN OUTCOME MEASURES: Bone marrow mesenchymal stem cells (BMSCs) were identified by flow cytometry; the expression of nestin, neuron-specific enolase, and glial fibrillary acidic protein were detected by immunocytochemistry. RESULTS: Flow cytometry showed that the cell surface markers CD90 and CD106 were positive and CD45 and CD34 were negative. Immunocytochemical detection showed that the Yiqi Huoxue Decoction group induced differentiated nestin cells at 1 and 3 h and induced neuron-specific enolase and glial fibrillary acidic protein cells that differentiated for 5 h, which was significantly different from the control group. (P<0.01). Conclusion: Yiqihuoxue decoction can induce the differentiation of bone marrow mesenchymal stem cells into neural cells in vitro.