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

Baicalin induces the differentiation of human umbilical cord blood mesenchymal stem cells into neuron-like cells in vitro

目的：体外诱导脐血间充质干细胞定向分化为神经元包括抗氧化剂法及细胞因子法,如何选择一种既能够如细胞因子般可广泛保护神经,又具有较强抗氧化作用的诱导剂？通过广泛筛选,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。方法：实验于2005-02/06在南昌大学第一附属医院神经内科实验室完成。①对象及药物：无内外科并发症和传染性疾病、无血液系统疾病、乙肝表面抗原阴性、非高危妊娠、年龄23-35岁的健康孕妇足月顺产儿的脐带血10份,由南昌大学第一附属医院产科提供,孕妇及其家属对治疗及实验均签署知情同意书，实验经医院医学伦理委员会批准。黄芩苷，分子量446，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。②实验方法：无菌条件下采集胎儿脐带血，肝素抗凝，用明胶沉降＋密度梯度离心两步法分离出脐血单个核细胞，经冷冻保存和复苏后，调整细胞浓度为1×10^9L^-1，以含20％胎牛血清、谷氨酰胺、B27、粒巨噬细胞集落刺激因子300μg/L、干细胞因子300μg/L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100μmol／L：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2mmol／L；共培养组添加黄芩苷100μmol／L、β-巯基乙醇2mmol／L。连续培养4周。③实验评估：观察细胞形态学变化；采用流式细胞仪检测间充质干细胞表面标志；制作细胞爬片，采用免疫细胞化学染色评价神经细胞特异性烯醇化酶、神经微管相关蛋白2、胶质纤维酸性蛋白阳性细胞表达率。结果：①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，且发出分支，形成圆锥状终末端。β-巯基乙醇组、共培养组细胞虽然也有上述类似形态学的改变，但少量细胞存在脱落、死亡现象，且随着培养时间延长呈逐渐增加的趋势。②细胞表型检测：扩增培养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。③细胞扩增培养过程中黄芩苷的预诱导效果：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），共培养组无明显差异（P〉0．05）。各组胶质纤维酸性蛋白阳性细胞表达率均低于1％。结论：100μmol／L黄芩苷可促进脐血间充质干细胞体外扩增，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。

AIM： Both the ways of antioxidant agent and cytokines can induce human umbilical cord blood mesenchymal stem cells （MSCs） to differentiate into neuron in vitro. But how to select an inductor, which has the ability of either neuro-protection like cytokines or powerful antioxidation, is a key now. After sieved extensively, this study investigated the effects of baicalin on in vitro purification, amplification and differentiation into neuron-like cells of human umbilical cord blood MSCs. METHODS： Experiments were performed at the Laboratory of Department of Neurology of First Affiliated Hospital of Nanchang University from February to June 2005. （1）Ten samples of cord blood were collected from healthy full-term normal delivery pregnant woman aged 23-35 years, who did not have the histories of complications and infectious disease, hematological system disease, positive hepatitis B virus surface antigen or high-risk pregnancy. The samples were provided by Department of Obstetrics of First Affiliated Hospital of Nanchang University. Informed consents were obtained from pregnant women and their family. The experiment was approved by Hospital＇s Medical Ethics Committee. Baicalin （446 molecular weight, 〉95% purity） was offered by Department of Pharmaceutical Preparation of Xiangya Medical College of Central South University. Beta-mercaptoethanol was bought from SABC. （2）Human cord blood was obtained sterilely with preservative-free heparin. Cord blood mononuclear cells were isolated by the two-step assay of gelatin sedimentation plus Ficoll centrifugation separation. After cryopreservation and resuscitation, cells were adjusted to 1 × 10^9 L^-1 and purified and expanded in DMEM liquid culture medium containing 20% fetal bovine serum, glutamine, B27, granulocyte colony-stimulating 300μ g/L, stem cell factor 300 μg/L. According to the kind of antioxidant, experiments were divided into four groups as follows, a baicalin group （100 μ mol/L baicalin）, a blank control group, a β-mercaptoethanol group （2 mmol/L β -mercaptoethanol） and the co-culture group （100 μ mol/L baicalin and 2 mmol/L 13 -mercaptoethanol）. （3）After four weeks, MSCs were observed. The surface antigen expression of MSCs was detected by flow cytometry. Four subgroups of MSCs were induced in the media. The expression of neuron specific enolase, microtubule associated protein 2 and glial fibrillary acidic protein was detected by immunocytochemistry. RESULTS： （1）Morphological observation： Cord blood mononuclear cells adhered to the wall 2-3 days after culture, and reached a peak 2 weeks later, formed a confluence of about 80%-90% 3 weeks after culture when the cord blood MSCs were mainly consisted of homogeneous long fusiform. Four weeks later, some fusiform cells in the baicalin group contracted with thin ecphyma, while some were round, cone, triangle, with several slender ecphyma that had branchs on the pseudopodia, forming cone-shape end. Except for above-mentioned appearances, a few cells defoliated and died, which became increased with time prolonged. （2）Four weeks after culture, cells were positive for CD45 in the blank control group, while cells in other groups were positive for CD29 and CD83, especially in the co-culture group, followed by baicalin group and β -mercaptoethanol group. There were significant differences in pairwise comparison （P 〈 0.01）. Otherwise, there were no CD34^＋ cells appeared in the four groups. （3）After culture for 4 weeks, the expression of neuron specific enolase and microtubule associated protein 2 were lower in the blank control group and β -mercaptoethanol group compared to the baicalin group （P 〈 0.01）, and no significant difference was found in the co-culture group （P 〉 0.05）. The percentage of cells expressed glial fibrillary acidic protein was lower than 1% in the four groups. CONCLUSION： 100 μ mol/L baicalin can promote in vitro amplification of cord blood MSCs, which can be greatly reinforced with the increase in antioxidation. Baicalin also can induce the differentiation of cord blood MSCs into neuron-like cells to some degree.