中药单体黄芩苷体外诱导人脐血间充质干细胞向神经元样细胞的分化

背景:抗氧化剂法及细胞因子法是体外诱导脐血间充质干细胞定向分化为神经元的2种方法,怎样选择既可广泛保护神经,又具有较强抗氧化作用的诱导剂?通过广泛筛选,应用中药下预脐血间充质干细胞的体外纯化、扩增并向神经元细胞分化.目的:观察中药单体黄芩苷对人脐血间充质干细胞体外纯化、扩增及向神经元样细胞诱导分化的作用.设计,时间,地点:随机对照,细胞学体外观察,于2005-02/06在南昌大学第一附属医院神经内科实验窒完成.材料:年龄为23～35岁的健康孕妇足月顺产儿的脐带血10份;黄芩苷,纯度＞95%,由中南大学湘雅医学院药剂科提供:抗氧化添加剂β-巯基乙醇为SABC公司产品.方法:采集胎儿脐带血,肝素抗凝,分离出脐血单个核细胞,调整细胞浓度为1×109L-1,以含体积分数为20%胎牛血清、谷氨酰胺、B27、粒巨噬细胞集落刺激因子、干细胞因子的DMEM液体培养基进行纯化和扩增.按照抗氧化添加剂的不同分为4组:黄芩苷组加入黄芩苷:空白对照组不添加抗氧化剂;β-巯基乙醇组添加β-巯基乙醇;共培养组添加黄芩苷、β-巯基乙醇.连续培养4周.主要观察指标:①脐血冷冻保存后7,14,21,28dCD34及CD29阳性细胞的表达.②细胞形态学观察.③流式细胞仪检测间充质干细胞表面标志.④免疫细胞化学染色观察间充质干细胞扩增培养4周后神经细胞特异性烯醇化酶、神经微管相关蛋白2和胶质纤维酸性蛋白阳性细胞的表达.结果:①与冻存前相比,冻存后7,14,21,28 d脐血单个核细胞锥虫蓝拒染率均明显降低(P＜0.05).②细胞形态学观察:脐血单个核细胞培养第2-3天开始贴壁生长,2周左右达高峰,3周后细胞生长达80%～90%融合,此时脐血原始间充质干细胞呈较均一的长梭形.4周后,黄芩苷组原来呈梭形的胞体一部分发生收缩,细胞边缘出现细的突起;一部分胞体逐渐近似圆形、锥形、三角形,伪足有多个细长突起.β-巯基乙醇组、共培养组有少量细胞存在脱落、死亡现象,且随着培养时间延长呈逐渐增加的趋势.⑧培养4周后,空白对照组以CD45阳性细胞为主;其余3组均以CD29和CD83阳性细胞为主,其中共培养组最多,黄芩苷组次之,β-巯基乙醇组最少,组间两两比较差异具有显著性意义(P＜0.01);各组贴壁生长细胞中均未见CD34阳性细胞.④扩增培养4周后与黄芩苷组比较,空白对照组、β-巯基乙醇组神经细胞特异性烯醇化酶及神经微管相关蛋白2阳性细胞表达率均显著降低(P＜0.01).各组胶质纤维酸性蛋白阳性细胞表达率均低于1%.结论:100 μ mol/L黄芩苷可促进脐血间充质下细胞体外扩增,且随抗氧化作用时间的延长效果显著增强,同时在一定程度上还能够诱导其向神经元样细胞方向分化.

Baicalin induces the differentiation of human umbilical cord blood-derived mesenchymal stem cells towards neurons-like cells in vitro

BACKGROUND: Both antioxidant and cytokine can induce the differentiation of umbilical cord blood-derived mesenchymal stem calls (UCB-MSCs) towards neuron-like cells in vitro. It remains unclear regarding how to select an inductor that has the ability of either neuro-protection like cytokines or powerful antioxidation. After wide screening, baicalin has been included in this study. OBJECTIVE: To observe the effects of baicalin on in vitro purification, amplification, and differentiation towards neuron-like cells of hUCB-MSCs.DESIGN, TIME AND SETTING: A randomized, controlled, cytological in vitro observation was performed at the laboratory of Department of Neurology, First Affiliated Hospital of Nanchang University between February and April 2005. MATERIALS: Ten portions of UCB were collected from healthy full-term normal delivery pregnant women aged 23-25 years old. Baicalin with purity > 95% was purchased from Department of Pharmaceutics, Xiangya Medical College of Central South University. Antioxidant additive J -mercaptoethanol was provided by Sino-American Biotechnology Company, China. METHODS: The collected UCB was anticoagulated with heparin to separate mononuclear cells. After concentration adjustment (1 ×109/L), UCB mononuclear cells were purified and amplified with dulbecco's modified eagle's medium containing fetal bovine serum (0.2 volume fraction), glutamine, B27, granulocyte colony-stimulating factors, and stem cell factors. According to antioxidant additive application, 4 groups were set: baicalin, blank control, β -mercaptoethanol, and baicaUn+ β -mercaptoethanol. Cells in each group were cultured for a total of 4 consecutive weeks. MAIN OUTCOME MEASURES: (1) Detection of CD 34 and CD 29 immunoreactive expression on days 7, 14, 21, and 28 after cryopreservation. (2) Cellular morphology observation. (3) Detection of surface antigen expression of MSCs by flow cytometry. (4) Detection of neuron-specific enolase (NSE), microtubule associated protein 2 (MAP-2), and glial fibrillary acidic protein (GFAP) expression after 4 weeks of culture by immunocytochemistry. RESULTS: O Compared with prior to cryopraservation, trypan blue exclusion rate of UCB-MSCs was significantly reduced on days 7, 14, 21, and 28 after cryopreservation (P < 0.05). (2) Morphological observation: UCB mononuclear cells adhered to the wall 2-3 days after culture, reached a peak level at 2 weeks, and formed a confluence of approximately 80%-90% 3 weeks after culture; at this time, all UCB-MSCs displayed a spindle shaped appearance. Four weeks later, in the baicalin group, some spindle-shaped UCB-MSCs began to present shrinkage, with slender processes on the cell edge, and some UCB-MSCs tended to be spherical-, conical-, and triangle-shaped appearance, with many slender processes on the pseudopodia. In the β-mercaptoethanol and baicalin+β -mercaptoethanol groups, an increasing number of cells defoliated and died with culture time in addition to above-mentioned appearances. (3) Four weeks after culture, cells were positive for CD45 in the blank control group, while cells in the remaining groups were positive for CD29 and CD 83, in particular in the baicalin+ β -mercaptoethanol group, followed by the baicalin group, and lastly the β -mercaptoethanol group. Significant difference in CD29 and CD 83 immunoreactivity exhibited between groups (P < 0.01). No CD34 immunoraactive calls were found in each group. (4) Four weeks after culture, NSE and MAP-2 immunoreactive expression was significantly lower in the blank control and β -mercaptoethanol groups than in the baicalin group (P < 0.01). The percentage of cells expressing GFAP was lower than 1% in each group. CONCLUSION: 100 μmol/L baicalin can promote the in vitro amplification of UCB-MSCs in a time-dependent manner and also can induce the differentiation of UCB-MSCs towards neuron-like cells in vitro to some extent.