环王巴明抑制sonic hedgehog信号通路对氧糖剥夺/再复氧损伤大鼠皮质神经干细胞增殖的影响  

Effects of Inhibiting Shh Signaling Pathway with Cyclopamine Pretreatment on Proliferation of Cortical Neural Stem Cells after Oxygen-Glucose Deprivation / Reoxygenation Injury in Rats

作　　者：成薇[1] 王莉[1] 余萍萍[1] 沈长波[1] 杨琴[1] 

机构地区：[1]重庆医科大学附属第一医院神经内科,重庆400016

出　　处：《中国药学杂志》2016年第8期625-629,共5页Chinese Pharmaceutical Journal

基　　金：国家自然科学基金面上项目(81071119);国家神经病学临床重点专科建设资助项目(卫办医政函[2012]649号)

摘　　要：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 effect of inhibitting sonic hedgehog（ Shh） signaling pathway with cyclopamine pretreatment on proliferation of rat cortical neural stem cells（ NSCs） after oxygen-glucose deprivation / reoxygenation（ OGD / R） injury in vitro. METHODS The suspended culture was used for the isolation and purification of NSCs in neonatal Sprague-Dawley（ SD） rats.The third passage NSCs for adherent culture were deprived oxygen and glucose for 150 min and recovered oxygen and glucose for 24 h.There were three groups,including normal,model and cyclopamine pretreatment groups. NSCs were identified with immunofluorescence. CCK-8 assay was used to examine cell viability. The proliferation of NSCs was measured with Brd U assay and flow cytometry cell cycle. Western blot was used to detect the protein expressions of Ptc-1,Smo and Gli-1. RESULTS There were high expression of nestin protein in suspended and adherent cultured cells. The cell vitalities in model and cyclopamine groups were decreased significantly compared with the normal group. Especially,there was less cell vitality in cyclopamine group（ P〈0. 05）. There was significantly increased for NSCs proliferation and upregulated for Ptc-1,Smo and Gli-1 proteins in the model group. On the contrary,compared with the model group,NSCs proliferation and the expressions of Ptc-1,Smo and Gli-1 proteins in cyclopamine group were significantly decreased（ P〈0. 05）. CONCLUSION Cyclopamine pretreatment can inhibit NSCs proliferation after OGD / R injury. The result suggests that Shh signaling may participate in the regulation of NSCs proliferation after injury.

关 键 词：环王巴明 预处理 神经干细胞 氧糖剥夺/再复氧 增殖 sonic HEDGEHOG 

分 类 号：R965[医药卫生—药理学]