摘要
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—40μmol／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、B一巯基乙醇、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；Hoechest 33 258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 study the possibility and mechanism by which the human umbilical blood mesenchymal stem cells （MSCs） are induced to differentiate into neuron in vitro by baicalin, a kind of flavonoid isolated from an important medicinal plant Scutellariae Radix. Methods Human cord blood was obtained viasterile procedure with 20 U/ml of preservative-free heparin. MSCs were isolated by the two-step assay of gelatin sedimentation plus Ficoll centrifugation separation, purified and amplified in the liquid culture medium. According to the kind of antioxidants, the experiment was conducted in five groups： induction group, control group Ⅰ , control group Ⅱ, control group Ⅲ and control group Ⅳ. Five subgroups of MSCs amplificated ex vivo for 2 weeks in each group were induced by the media containing baicalin （ BC, 200 -400μmol/L） or balcalin-free for seven days. The media consisted of induction medium （DMEM plus 200-400μmol/L of baicalin） and post-induction medium （DMEM plus 200 -400μmol/L of baicalin, B27）. The expression of neuronal or glia specific markers was evaluated by using indirect immunofluorescence cytochemistry staining. The percentage of differentiated cells and living cells was measured by Hoechest 33 258 staining assay. Results After induction for 7 days, MSCs displayed neuronal morphologies, such as pyramidal cell bodies and processes formed extensive network. The undifferentiated cells did not exhibit a neuronal or glial phenotype, while the differentiating cells expressed NSE and MAP-2, the specific markers of neuron, but did not express GFAP, the specific marker of glia on the seventh day after induced by baicalin. The percentage of NSE and MAP-2 expressed on the seventh day after induced by baicalin was （76.3 ± 9.2） % and （78.5 ± 5.5 ） % , respectively, which was significantly higher compared with control group Ⅰ, control group Ⅱ and control group Ⅲ （P 〈0.01）, respectively. In addition, the ratio of living cells after induced for seven days in the BC group was （ 85.3 ± 4.8） %, which increased significantly compared with control group Ⅰ , control group Ⅱ and control group Ⅲ （ P 〈 0. 01 ）, respectively. Conclusions Baicalin may induce the umbilical blood MSCs to neuron or neuron-alike cells in vitro in a moderate and stable manner. The mechanism of such an induction may be related with its controlling the activity of NF-κB which regulates the production of many kinds of cytokines, such as brainderived neurotrophic factor （BDNF）, etc.
出处
《中华儿科杂志》
CAS
CSCD
北大核心
2006年第3期214-219,共6页
Chinese Journal of Pediatrics
关键词
黄芩甙
胎血
间充质干细胞
诱导分化
神经元
Baicalin
Human umbilical cord blood
Mesenchymal stem cells
Differentiation
Neuron
