益气活血方体外诱导大鼠骨髓间充质干细胞向神经细胞的分化

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1 200g,赤芍60g,地龙30 g,红花30g,再加入920mL.水,煎煮1h,滤过,药渣再加入8 640mL.水,煎煮1h,滤过,合并2次煎液,浓缩至600 mL.,加入挥发油及吐温-80 3 mL,混匀,装瓶进行高压高温消毒.每毫升相当于生药2.4 g.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40 mm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24 h,益气活血方组而后换为含诱导液二甲业砜、丁羟茼醚、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、胶质纤维酸性蛋向的表达.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.

Yiqihuoxue recipe induces differentiation of rat bone marrow mesenchymal stem cells towards neurons in vitro

BACKGROUND: Previous studies have demonstrated that Yiqihuoxue recipe (Buyanghuanwu decoction) can inhibit cell apoptosis and antagonize free radical injury in cerebral ischemia/reperfusion models. However, there have been few reports regarding the effects of Yiqihuoxue recipe on stem cell differentiation. OBJECTIVE: To observe the effects of Yiqihuoxue recipe on the differentiation of rat bone marrow mesenchymal stem cells (MSCs) towards neurons. DESIGN, TIME AND SETTING: The present controlled observational expedment based on cells was performed at the Henan University of Traditional Chinese Medicine, i.e., the Third-Grade Laboratory of the State Administration of Traditional Chinese Medicine between March 2006 and February 2008.MATERIALS: Twenty clean Sprague-Dawley (SD) rats, aged 1-2 months, weighing 100-150 g, of either gender, were included in this study. The Chinese medicine compound Yiqihuoxue recipe was made in the Manufacturing Laboratory of the First Affiliated Hospital of Henan College of Traditional Chinese Medicine as follows. 60 g Danggui (Radix Angelicae Sinensis), 30 g Taoren(Semen Persicae), 30 g Chuanxiong(Szechwan Lovage Rhizome) were selected and mixed in 720 mL water for extract volatile oil for later use. The remaining gruffs and extract were supplemented with 1 200 g Huangqi(Radix Astragali), 60 g Chishao (Radix Paeoniae Rubra), 30 g Dilong(Lumbricus), 30 g Honghua(Flos Carthami) before 920 mL water was added. After I hour of decoction and filtering, the gruffs were decocted for another 1 hour after adding 8 640 mL water. Then all decocted liquid was merged and condensed to 600 mL and mixed with 3 mL of volatile oil and tween-80. After high-temperature and high-pressure stedlization, the products were preserved for future use. 1 mL of drug was equal to 2.4 g raw herbs. METHODS: Bone marrow mononuclear cells were isolated by density gradient centrifugation. After culture, bone marrow MSCs were amplified and purified. Passage 10 cell suspension was inoculated into a 40 mm-diameter plastic culture dish. Dulbecco's modified eagle's medium supplemented with 0.1 volume fraction of fetal bovine serum and basic fibroblast growth factors was added for 24 hours of culture when adherent cells reached 60%-90% confluence in each group. Thereafter, the Yiqihuoxue group was incubated for 5 hours using medium containing DMSO, butylated hydroxyanisole, 13-mercaptoethanol, and Yiqihuoxue recipe; simultaneously, the control group was treated for 5 hours with fluid containing DMSO, butylated hydroxyanisole, and β- mercaptoethanol.MAIN OUTCOME MEASURES: Identification of bone marrow MSCs by flow cytometry; Detection of Nestin-, and neuron specific enolase(NSE)-, and glial fibrillary acidic protein(GFAP)-positive expression by immunohistochemistry. RESULTS: Flow cytometery results demonstrated that cell surface marker CD90 and CD106 expression was positive, while CD45 and CD34 expression was negative. Immunohistochemistry results showed that the numbers of Nestin-positive cells (1 and 3 hours after induction) and NSE- and GFAP-positive cells (5 hours after induction) were significantly greater in the Yiqihuoxue group than in the control group (P < 0.01). CONCLUSION: Yiqihuoxue recipe can induce the differentiation of bone marrow MSCs towards neurons in vitro.