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目的探讨中药黄芩苷体外诱导人脐血间充质干细胞(MSCs)向神经元样细胞分化的可行性及其可能的机制。方法采集健康孕妇足月顺产儿的脐带血,共5人份,以肝素抗凝,采用明胶沉降加密度梯度离心两步法分离脐血单个核细胞,加入含黄芩苷50μmol/L的液体培养体系中进行扩增培养。取黄芩苷体外扩增2周的人脐血MSCs,采用黄芩苷诱导24h后,继续维持诱导6d,诱导30min后开始在倒置显微镜下动态观察脐血MSCs生长情况及诱导前后形态学变化。免疫细胞化学染色评价神经细胞特异性烯醇化酶(NSE),微管相关蛋白2(MAP-2)阳性细胞的表达。诱导液(DMEM培养基,200~400μmol/L黄芩苷),37℃,5%CO2诱导24h。维持诱导液(DMEM培养基,200~400μmol/L黄芩苷,B27)继续维持诱导1周。实验共分4组,分别为诱导组;对照1组(诱导液和维持液均不含黄芩苷)、对照2组(诱导液和维持液含3mmol/L的β-巯基乙醇,不含黄芩苷)、对照3组(诱导液和维持液含20g/L二甲基亚砜和20mmol/L丁化羟基苯甲醚,不含黄芩苷)、对照4组(诱导液和维持液均含有上述浓度的黄芩苷、β-巯基乙醇、二甲基亚砜和丁化羟基苯甲醚),各组分别在诱导6h、24h、7d留取标本,制作细胞爬片,细胞固定后,免疫细胞化学染色评价NSE和MAP-2阳性细胞的表达率;Hoechest33258染色,评价细胞存活率。结果诱导组诱导6h后,细胞微丝收缩,原来梭形的脐血MSCs胞体已发生收缩,细胞边缘变得不规整,出现了细的突起。7d后多数细胞成锥形,交织成网,形成较典型的神经元细胞样形态结构,免疫细胞化学染色显示黄芩苷诱导组NSE、MAP-2阳性细胞表达率以及细胞存活率分别为(76.3±9.2)%、(78.5±5.5)%、(85.3±4.8)%,显著高于对照1、2、3组(P<0.01),分别为(4.6±0.6)%、(0.7±0.6)%、(46.7±9.2)%;(63.3±6.8)%、(40.9±5.1)%、(66.5±5.2)%和(71.6±4.7)%、(42.3±4.5)%、(72.8±7.6)%。此外,各组胶质纤维酸性蛋白(GFAP)的表达率均低于1%。结论低浓度的黄芩苷体外扩增2周后的MSCs中已有少量表达NSE的阳性细胞出现,这在一定程度上黄芩苷起到了预诱导分化作用;黄芩苷能够体外诱导脐血MSCs分化为神经元样细胞,诱导过程中黄芩苷的诱导作用温和、稳定而持久;诱导出的神经元样细胞成活时间长,其诱导机制可能与黄芩苷的抗氧化、调控细胞NF-κB的活性从而刺激多种细胞因子的表达和生成有关。
Objective To investigate the feasibility and possible mechanism of baicalin-induced differentiation of human umbilical cord blood mesenchymal stem cells (MSCs) into neuron-like cells in vitro. Methods Umbilical cord blood of healthy pregnant women was collected from term infants, and a total of five patients were included. Anticoagulation with heparin was performed. Umbilical cord blood mononuclear cells were isolated by gelatin sedimentation plus density gradient centrifugation, and a liquid culture system containing baicalin 50 μmol/L was added. In the amplification culture. The human umbilical cord blood MSCs expanded from baicalin in vitro for 2 weeks were induced with baicalin for 24 hours and continued to be induced for 6 days. After 30 minutes of induction, the growth of umbilical cord blood MSCs and the morphological changes before and after induction were observed under inverted microscope. Immunocytochemical staining was used to evaluate the expression of neuron-specific enolase (NSE) and microtubule-associated protein 2 (MAP-2) positive cells. Inducing solution (DMEM medium, 200-400 μmol/L baicalin), 37°C, 5% CO2 for 24 h. The induction fluid (DMEM medium, 200-400 μmol/L baicalin, B27) was maintained for 1 week. The experiment was divided into 4 groups: the induction group; the control group 1 (no baicalin in the induction and maintenance fluids) and the control 2 group (the induction and maintenance fluid contained 3 mmol/L of β-mercaptoethanol and no baicalin Control group 3 (Inducer and maintenance fluids contain 20 g/L dimethyl sulfoxide and 20 mmol/L butylated hydroxyanisole, no baicalin), and control group 4 (induction and maintenance fluids contain the above concentrations) Baicalin, β-mercaptoethanol, dimethyl sulfoxide and butylated hydroxyanisole) were collected from each group at 6 h, 24 h, and 7 d after induction, and cells were made into slides. After cell fixation, immunocytochemical staining was performed. The expression rate of NSE and MAP-2 positive cells was evaluated; Hoechst 33258 staining was performed to evaluate cell viability. Results After induction for 6 h, the microfilaments of the cells in the induction group contracted, and the cell bodies of the original shuttle umbilical cord blood MSCs contracted. The edges of the cells became irregular and fine protrusions appeared. After 7 days, most of the cells became conical and interwoven to form a more typical neuronal cell-like morphological structure. Immunocytochemical staining showed that the expression rate of NSE and MAP-2 positive cells and the cell survival rate in the baicalin-induced group were (76.3±). 9.2)%, (78.5±5.5)%, (85.3±4.8)%, significantly higher than the control 1, 2 and 3 groups (P<0.01), (4.6±0.6)%, (0.7±0.6)%, respectively (46.7±9.2)%; (63.3±6.8)%, (40.9±5.1)%, (66.5±5.2)%, and (71.6±4.7)%, (42.3±4.5)%, (72.8±7.6)%. In addition, the expression of glial fibrillary acidic protein (GFAP) in each group was less than 1%. Conclusion A small amount of NSE-expressing positive cells appeared in MSCs with low concentration of baicalin after 2 weeks of in vitro expansion. In a certain extent, baicalin can play a role in pre-differentiation. Baicalin can induce differentiation of umbilical cord blood MSCs into nerves in vitro. In the cell-like induction process, baicalin induction is mild, stable, and long-lasting; the induced neuron-like cells survive for a long time, and its induction mechanism may be related to the antioxidation of baicalin, which regulates the activity of NF-κB, thereby stimulating more The expression and production of several cytokines are related.