慢病毒介导的BAG-1L基因对缺氧状态下人神经母细胞瘤细胞的保护作用  

Protective effect of BAG-IL mediated by lentivirus in human neuroblastoma cells induced by hypoxia

作　　者：王芸[1] 李庆淑 贾超[2] 谢春雨[1] 宋砚坤 张楠[1] 曲彦[2] Wang Yun Li Qingshu Jia Chao Xie Chunyu Song Yankun Zhang Nan Qu Yan(School of Nursing, Medical College of Qingdao University, Qingdao 266023, Shandong, China Department of Critical Care Medicine, Affiliated Qingdao Municipal Hospital of Qingdao University, Qingdao 266071, Shandong, China)

机构地区：[1]青岛大学医学部护理学院,山东青岛266023 [2]青岛大学附属青岛市市立医院东院重症医学科,山东青岛266071

出　　处：《中华危重病急救医学》2017年第3期239-243,共5页Chinese Critical Care Medicine

基　　金：国家自然科学基金（81571938,81501706）

摘　　要：目的探讨慢病毒介导Bcl-2结合抗凋亡基因1L（BAG-1L）过表达对缺氧／复氧诱导人神经母细胞瘤细胞（SH-SY5Y）损伤的保护作用，研究其对磷酸肌醇-3激酶／丝氨酸-苏氨酸蛋白激酶（PI3K/AKT）通路的影响。方法体外培养SH-SY5Y细胞，取对数生长期细胞分为重组慢病毒感染组（感染携带BAG-1L基因及荧光蛋白基因的重组慢病毒）、载体对照组（感染携带荧光蛋白基因但不携带BAG-1L基因的慢病毒）及细胞对照组（未感染慢病毒）。感染48h后采用蛋白质免疫印迹试验（Western Blot）检测靶细胞BAG-1L的表达；3组细胞经缺氧8h／复氧24h处理后，采用CCK-8细胞增殖及细胞毒性检测试剂盒检测细胞活性；采用别藻蓝蛋白标记的膜联蛋白V／7-氨基放线菌素D（Annexin V-APC／7-AAD）双染法检测细胞凋亡，并进行流式分析；采用Western Blot试验检测细胞BAG-1L、热休克蛋白70（HSP70）、AKT及磷酸化AKT（p-AKT）的蛋白表达。结果重组慢病毒感染48h后可观察到外源性BAG-1L特异性蛋白条带，而细胞对照组和载体对照组无此特异性蛋白条带。经缺氧，复氧处理后，重组慢病毒感染组细胞活性较细胞对照组和载体对照组均明显增高（A值：0．689±0．036比0．425±0．013、0．400±0．012），细胞凋亡明显减少[凋亡率：（26．97±1．82）％比（36．60±1．45）％、（35．77±3．74）％]，BAG-1L、HSPT0、P-AKT蛋白表达水平明显升高（BAG-1L蛋白（灰度值）：2．405±0．167比0．529±0．141、0．601±0．099，HSP70蛋白（灰度值）：0．997±0．123比0．634±0．091、0．584±0．106，p-AKT蛋白（灰度值）：1．234±0．118比0．661±0．210、0．712±0．199，均P〈0．01]；而AKT蛋白表达虽高于细胞对照组和载体对照组（灰度值：1．103±0．269比0．646±0．188、0.791±0．326），但差异无统计学意义（均P〉O．05）。细胞对照组和载体对照组各指Objective To investigate the protective effects of lentivirus mediated Bcl-2-associated athanogene 1L （BAG-1L） over-expression on human neuroblastoma cells （SH-SYSY） induced by hypoxia/re-oxygenation, and to study its effect on the phosphoinositide 3 kinase serine/threonine protein kinase （PI3K/AKT） pathway. Methods SH-SYSY cells were cultured in vitro, and the cells at logarithmic phase were collected, and they were divided into recombined lentiviral infection group [infected by lentivirus containing BAG-1L and green fluorescent protein （GFP） gene], vector control group （infected by lentivirus containing GFP without BAG-1L gene） and cell control group （non-infection）. Western Blot was used to detect the expression of BAG-1L in target ceils after infection for 48 hours. SH-SY5Y cells were subjected to hypoxia for 8 hours and re-oxygenation for 24 hours, then the cell counting kit-8 （CCK-8） was used to detect the cell activity, and the apoptosis was detected by flow cytometry after allophycocyanin labeled annexin V/7-amino actinomycin D （Annexin V-APC/7-AAD） staining. Western Blot was used to detect the protein expressions of BAG-1L, heat shock protein 70 （HSP70）, AKT and phosphorylated AKT （p-AKT）. Results After infection for 48 hours, exogenous BAG-1L protein bands were observed in recombined lentiviral infection group, but not observed in cell control group and vector control group. After hypoxia/re-oxygenation treatment, the cell viability in recombined lentiviral infection group was significantly higher than that in cell control group and vector control group （A value： 0.689 ± 0.036 vs. 0.425 ± 0.013, 0.400 ± 0.012）, apoptosis was significantly decreased [apoptosis rate： （26.97 ± 1.82）% vs. （36.60±1.45）%, （35.77 ± 3.74）%], the protein levels of BAG-1L, HSP70 and p-AKT were significantly increased [BAG-1L protein （gray value）： 2.405±0.167 vs. 0.529±0.141, 0.601±0.099; HSP70 protein （gray value）： 0.997±0.123 vs. 0.634±0.091, 0.58

关 键 词：Bcl-2结合抗凋亡基因 神经细胞 缺氧/复氧 抗凋亡 P13K/AKT通路 

分 类 号：R743[医药卫生—神经病学与精神病学]