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目的探讨环王巴明预处理抑制sonic hedgehog信号通路对体外氧糖剥夺/再复氧(oxygen-glucose deprivation/reoxygenation,OGD/R)损伤大鼠大脑皮质神经干细胞(neural stem cells,NSCs)增殖的影响。方法新生SD大鼠大脑皮质神经干细胞采用悬浮培养法分离纯化。第3代神经干细胞贴壁培养后氧糖剥夺150 min,复氧培养24 h。实验分为正常组、模型组及环王巴明预处理组。细胞鉴定采用免疫荧光法,细胞活力采用CCK-8法检测,细胞增殖采用Brd U法及流式细胞周期检测,Ptc-1、Smo、Gli-1蛋白表达采用Western blot检测。结果悬浮及贴壁培养细胞均高表达巢蛋白。模型组及环王巴明组细胞活力较正常组显著降低。其中,环王巴明组降低更明显(P<0.05)。模型组细胞增殖明显,Ptc-1、Smo、Gli-1蛋白表达上调,而环王巴明组细胞增殖较模型组低,Ptc-1、Smo、Gli-1蛋白表达下调(P<0.05)。结论环王巴明预处理能抑制体外氧糖剥夺/再复氧损伤后神经干细胞的增殖,提示Shh信号可能参与损伤后神经干细胞增殖的调控。
Objective To investigate the effects of cyclopentamine pretreatment on proliferation of neural stem cells (NSCs) in rat cerebral cortex after oxygen-glucose deprivation / reoxygenation (OGD / R) injury in vitro influences. Methods The neural stem cells of neonatal SD rats were isolated and purified by suspension culture. The third generation of neural stem cells adherent culture after oxygen deprivation 150 min, reoxygenation culture 24 h. The experiment was divided into normal group, model group and Central Plains pretreatment group. The cells were identified by immunofluorescence method. Cell viability was detected by CCK-8 assay. Cell proliferation was detected by BrdU assay and flow cytometry. The protein expressions of Ptc-1, Smo and Gli-1 were detected by Western blot. Results Nestin was highly expressed in both suspension and adherent cells. The viability of model group and ring Wang Baming group was significantly lower than that of the normal group. Among them, the reduction of cyclopentamine group was more obvious (P <0.05). The expression of Ptc-1, Smo and Gli-1 were up-regulated in the model group, while the expression of Ptc-1, Smo and Gli-1 was down-regulated in the cyclopentamine group compared with the model group (P <0.05). Conclusion Pretreatment with cyclopentamine can inhibit the proliferation of neural stem cells in vitro after oxygen glucose deprivation / reoxygenation injury, suggesting that Shh signaling may be involved in the regulation of neural stem cell proliferation after injury.