脑康Ⅱ号对新生大鼠海马神经干细胞增殖分化的影响

Effect of Naokangerhao （脑康Ⅱ号） on proliferation and differentiation of hippocampal neural stem cells of newborn rats

目的探讨脑康Ⅱ号含药血清对体外培养大鼠海马神经干细胞（NSCs）增殖分化作用的影响。方法用无血清DMEM／F12[含20μg／L碱性成纤维细胞生长因子（bFGF）和表皮生长因子（EGF）及B27]体外培养新生Wistar大鼠海马NSCs，在NSCs分化过程中添加大、中、小剂量（分别为2．0、1．0、0．5kg／L）脑康I号含药血清进行干预，以免疫荧光及细胞化学方法对NSCs及其分化后的细胞进行鉴定。结果从新生大鼠海马分离培养的NSCs能够表达巢蛋白，并具有分化为神经元和星形胶质细胞的能力；5d时，脑康I号各剂量组神经球突起长度均显著长于对照组（P〈0．05或P〈O．01）；与对照组比较，脑康Ⅱ号各剂量组细胞分化为神经元或星形胶质细胞率均明显升高（P〈0．05或P〈0．01）。结论脑康Ⅱ号可促进NSCs向神经元方向分化，并对所分化的神经元样细胞有促生长、发育作用。

Objective To observe the effect of Naokangerhao （NKEH, 脑康Ⅱ号） serum on proliferation and differentiation of hippocampal neural stem cells （NSCs） of newborn rats in vitro. Methods Hippocampal NSCs from Wistar rats that were born in 24 hours were isolated and cultivated in vitro to undergo clone culturing; serum free DMEM/F12, 20 μg/L basic fibroblast growth factor （bFGF）, 20 μg/L epidermal growth factor （EGF） and B27 were used in the culture. During the progress of NSCs differentiation, three different doses of NKEH （2.0, 1.0 and 0.5 kg/L） in serum were added for interference, and immunofluorescence and immunocytochemistry stain were applied to identify the cells in the proliferation and differentiation. Results The neural spheres obtained from cultured hippocampal NSCs of newborn rats were positive for nestin expression, with the potential for further cloning and capability of differentiating into neurons or glial cells. On the 5th day of cultivation, the neurite length of neural sphere in the three NKEH groups was significantly longer than that in the control group （P 〈0.05 or P 〈0.01）. Results from immunofluorescence showed that the percentages of β-Ⅲ-tubulin and gliofibrillary acid protein （GFAP） positive cells differentiated from NSCs in different NKEH groups were much higher than those in control group （P〈0. 05 or P〈 0. 01）. Conclusion NKEH can promote the differentiation of NSCs into nerve cell and facilitate the growth and development of differentiated neuron-like cells.