摘要
目的:探讨右美托咪定(DEX)减轻皮质酮(CORT)抑制神经干细胞(NSCs)的增殖作用及其可能的作用机制。方法:取孕15 d SD大鼠,分离胎鼠大脑皮质培养原代NSCs,进行NSCs标志蛋白神经上皮干细胞蛋白(Nestin)和胚胎干细胞关键蛋白(SOX2)染色鉴定;RT-PCR法检测肾上腺α-2受体各亚型α-2A受体、α-2B受体及α-2C受体在NSCs上的表达;将NSCs分为Control组、DMSO组、CORT组(CORT 1μmol/L孵育24 h)和DEX组(DEX 0.01、0.1、1μmol/L预处理1 h后,再加入CORT继续孵育24 h)。LDH法检测NSCs的细胞毒性;EdU法检测细胞增殖能力;Western blot检测NSCs GSK-3β/β-catenin通路GSK-3β、p-GSK-3β、β-catenin和Cyclin D1表达水平。结果:分离的原代细胞经Nestin蛋白和SOX2蛋白染色鉴定NSCs的纯度超过97%;RTPCR结果显示肾上腺α-2受体的3种亚型在NSCs上均有表达;LDH法显示各组之间LDH的释放量差异无统计学意义。EdU法显示与Control组比,CORT组的NSCs增殖能力显著被抑制(P<0.05);与CORT组相比,当DEX剂量增加至1μmol/L时,NSCs的增殖能力有所恢复(P<0.05)。Western blot结果表明与Control组、DMSO组比,CORT组p-GSK-3β、β-catenin、Cyclin D1蛋白的相对表达量显著下降(P<0.05),与CORT组比,1μmol/L DEX预处理后,p-GSK-3β、β-catenin、Cyclin D1蛋白的相对表达量有所提升(P<0.05),Total GSK-3β蛋白在各组间的相对表达量没有明显变化;PI3K磷酸化抑制剂LY294002拮抗DEX对NSCs增殖的保护作用(P<0.05)。结论:DEX可以减轻CORT抑制NSCs的增殖作用,其机制与GSK-3β/β-catenin信号通路有关。
Objective:To investigate the alleviating effect of dexmedetomidine(DEX)on the proliferation of neural stem cells(NSCs)from corticosterone(CORT)-induced injury and explore the mechanisms potentially involved.Methods:NSCs were harvested from E15 SD rat brain and cultured.Neuroepithelial stem cell protein(nestin and SOX2)staining was identified.Whether NSCs express mRNA of theα-2A,α-2B,α-2C adrenoceptor subtypes was detected by Reverse transcriptase-PCR.The NSCs were randomly divided as the Control,DMSO,CORT(0.01,0.1,1μmol/L),Dexmedetomidine with CORT.The NSCs in the Dexmedetomidine with CORT group were pretreated with different concentrations of dexmedetomidine(0.01,0.1,1μmol/L)for 1 h prior to incubation with 1μmol/L CORT for 24 h.Cell cytotoxicity and Edu assays were used to figure out the optimal concentration of dexmedetomidine which can be used for the protein studies;Protein levels of GSK-3β,p-GSK-3β,β-catenin and Cyclin D1 were quantified with Western blot.Results:More than 98%of them were NESTIN/SOX2 positive. The three subtypes of α-2 receptor were expressed on NSCs. There was no significant difference between each group in the release of LDH. CORT significantly decreased NSCs proliferation, however, 1 μmol/L DEX increased NSCs proliferation. CORT inhibited p-GSK-3β, β-catenin and Cyclin D1 expression compared with DMSO group, but 1 μmol/L DEX pretreatment enhanced p-GSK-3β, β-catenin, Cyclin D1 expression,compared with CORT group. Total GSK-3β levels in each group did not change significantly. The protective effect of dexmedetomidine on CORT-exposed NSCs could be partly reversed by PI3K phosphorylation inhibitor LY294002. Conclusion: Dexmedetomidine may protect NSCs from corticosterone-induced injury via the GSK-3β/β-catenin signaling pathway.
作者
潘一钊
马君梅
黄坚坚
胡志妍
梅虹霞
林函
PAN Yizhao;MA Junmei;HUANG Jianjian;HU Zhiyan;MEI Hongxia;LIN Han(Department of Anesthesiology,Critical Care and Pain Medicine,the Second Affiliated Hospital&Yuying Children’s Hospital of Wenzhou Medical University,Wenzhou 325027,China;Zhejiang Province Key Lab of Anesthesiology,the Second Affiliated Hospital&Yuying Children’s Hospital of Wenzhou Medical University,Wenzhou 325027,China)
出处
《温州医科大学学报》
CAS
2020年第2期92-97,共6页
Journal of Wenzhou Medical University
基金
浙江省自然科学基金资助项目（LY14H090015）
关键词
皮质酮
右美托咪定
神经干细胞
细胞增殖
corticosterone
dexmedetomidine
neural stem cells
cell proliferation
