单磷酸腺苷激活的蛋白激酶在罗哌卡因致SH-SY5Y细胞凋亡中的作用  

Is activation of AMP-activated protein kinase involved in ropivacaine-induced SH-SY5Y cell apoptosis？

作　　者：周树勤[1] 卢俊[1] 张庆国[1] 许睿[1] 赖露颖[1] 徐世元[1] 

机构地区：[1]南方医科大学珠江医院麻醉科,广州市510282

出　　处：《中华麻醉学杂志》2011年第2期197-200,共4页Chinese Journal of Anesthesiology

基　　金：国家自然科学基金（30972843）

摘　　要：目的 评价单磷酸腺苷激活的蛋白激酶（AMPK）在罗哌卡因致神经母细胞瘤SH-SY5Y 细胞凋亡中的作用.方法 SH-SY5Y细胞在含15%胎牛血清的DMEM/F12培养基中于37℃、5%CO2细胞培养箱中培养.将SH-SY5Y细胞按5×105个/孔接种于6孔培养板中.采用随机数字表法,将培养孔随机分为5组：对照组（Ⅰ组）、空载体质粒pEGFP-N1组（Ⅱ组）、重组质粒pEGFP-N1-AMPKα2组（Ⅲ组）、阴性对照质粒pGPU6/GFP/Neo NC组（Ⅳ组）和重组质粒pGPU6/GFP/Neo-shRNA AMPKα2组（Ⅴ组）.Ⅰ组细胞不给予任何处理,其余各组按每孔4 μg质粒、10μl脂质体Lipofectamine2000转染SHSY5Y细胞.采用Western blot法测定AMPKα2蛋白表达水平.SH-SY5Y细胞经3 mmol/L罗哌卡因处理后,采用流式细胞术检测细胞内活性氧簇（ROS）水平;采用MTT法检测细胞存活情况,计算细胞存活率;分别采用流式细胞术和Hoechst33258染色检测细胞凋亡情况,计算细胞凋亡率.结果 重组质粒pEGFP-N1-AMPKα2可上调AMPKα2蛋白表达;而重组质粒pGPU6/GFP/Neo-shRNA AMPKα2可下调AMPKα2蛋白表达（P〈0.01）.下调AMPKα2蛋白表达可抑制细胞内ROS的生成,抑制细胞凋亡;而上调AMPKα2蛋白表达可促进细胞内ROS的生成,导致细胞凋亡（P〈0.01）.结论 罗哌卡因可通过激活AMPK引起SH-SY5Y细胞ROS生成增多,从而导致细胞凋亡.Objective To determine if activation of AMP-activated protein kinase （AMPK） is involved in ropivacaine-induced reactive oxygen species （ROS） production and apoptosis in human neuroblastoma cell line SHSY5Y.Methods SH-SY5Y cell line was purchased from cell center of Shanghai life Science Research Institute,Chinese Academy of Sciences and cultured in DMEM/F12 liquid culture medium containing 15 % bovine calf serum at 37 ℃ in incubator filled with 5% CO2 . Plasmids pGPU6/GFP/Neo-shRNA AMPKα2 and pEGFP-N1-AMPKα2were transfected into the SH-SY5Y cell line. The expression of AMPKα2 was determined by Western blot analysis.The SH-SY5Y cells transfected with recombinant plasmids were exposed to 3 mol/L ropivacaine. Intracellular ROS was detected by flow cytometry. Cell viability was quantitatively determined by MTT colorimetry assay. Apoptosis was assessed by flow cytometry and Hoechst33258 staining. Results The plasmid pEGFP-N1-AMPKα2 upregulated while pGPU6/GFP/Neo-shRNA AMPKα2 down-regulated the expression of AMPKα2 （ P 〈 0.01）. Down-regulation of AMPKα2 expression attenuated while up-regulation of. AMPKα2 expression promoted intracellular ROS production and cell apoptosis induced by ropivacaine （ P 〈 0.01） . Conclusion AMPK probably mediates ROS production and cell apoptosis induced by ropivacaine.

关 键 词：蛋白激酶类 酰胺类 细胞凋亡 

分 类 号：R965[医药卫生—药理学]