苯甲酸雌二醇对大鼠海马神经干细胞增殖分化的影响

Effects of estradiol benzoate on proliferation and differentiation of in vitro cultured hippocampal neural stem cells

目的研究苯甲酸雌二醇对大鼠海马神经干细胞增殖、分化的影响及其可能机制。方法取SD大鼠海马组织，分离神经干细胞后培养，通过Nestin免疫荧光染色鉴定神经干细胞。用不同浓度的苯甲酸雌二醇（10^-10、10^-9、10^-8、10^-7、10^-6mol／L）处理细胞，MTT方法检测神经干细胞的增殖，流式细胞术检测分化细胞中神经元和星形胶质细胞的比例。通过免疫印迹方法检测神经干细胞中蛋白表达情况。结果（1）海马神经干细胞呈神经球聚集生长，Nestin染色为阳性。海马神经干细胞内表达雌激素受体a（ERa）和雌激素受体p（ERp）；（2）与对照组相比，10μmol／L苯甲酸雌二醇处理之后，细胞活性明显升高（t=5．36，P=0．003），并且神经元特异性核蛋白（NeuN）阳性细胞所占比例升高（t=4．32，P=0．02），而神经胶质纤维酸性蛋白（GFAP）细胞阳性的比例下降（t=4．65，P=0．008），神经胶质源神经营养因子（GDNF）、胶质源神经营养因子受体al（GFRal）、转染重排蛋白（Ret）的表达增强。结论合适浓度的苯甲酸雌二醇能够促进体外培养的大鼠海马神经干细胞的增殖和分化，提高神经元的分化比例。

Objective To explore the potential mechanism of action of estradiol benzoate on proliferation and differentiation of rat hippocampal neural stem cells. Methods Hippocampus tissues from SD rats were dissociated and plated into culture flasks. Neural stem cells were identified by immunofluorescence against Nestin. After treatment with different concentration of estradiol benzoate （10^-10、10^-9、10^-8、10^-7、10^-6mol/L）,the proliferation and differentiation of neural stem cells were assessed by MTT assay and the proportion of neurons and astrocytes in differentiated cells was assessed by flow cytometry. Western blot assay was performed to detect the expression levels of protein in neural stem cells. Results （1） The neural stem cells formed neurospheres and grew in floating, and were Nestin-positive. Estrogen receptors, including ERα and ERβ, were expressed in neural stem cells;（2）After treatment with 10^-8 mol/L estradiol benzoate versus in the control group, the cell viability was significantly increased （t = 5.36, P = 0. 003）, and the ratio of neuron-specific nuclear protein（NeuN）-positive neuron cells in total cells was significantly inereased（t = 4.32, P = 0.02）, but the ratio of glial fibers acidic protein（GFAP）-positive cell in total cells was decreased（t = 4.65,P=0. 008）. The expression levels of GDNF, GFRal and Ret proteins were enhanced in the estradiol benzoate group compared with the control group. Conclusions Appropriate concentration of estradiol benzoate promotes proliferation and differentiation ofin vitrocultured hippocampal neural stem cells and elevates the ratio between neurons and glial cells differentiated from neural stem cells.