中性粒细胞明胶酶相关脂质运载蛋白对缺氧/复氧肾小管上皮HK-2细胞自噬作用的影响  

Effects of neutrophil gelatinase-associated lipocalin on autophagy of kidney tubules epithelial cell line HK-2 during hypoxia /reoxygenation

作　　者：张文静[1] 崔丽艳[1] 杨硕[1] 张捷[1] 

机构地区：[1]北京大学第三医院检验科,北京100191

出　　处：《临床检验杂志》2013年第10期765-768,共4页Chinese Journal of Clinical Laboratory Science

基　　金：国家自然科学基金面上项目(81271903);国家自然科学基金青年基金项目(61201093);国家科技支撑计划课题(2012BA137B01)

摘　　要：目的观察中性粒细胞明胶酶相关脂质运载蛋白(NGAL)对缺氧/复氧肾小管上皮细胞HK-2自噬水平的影响,探讨NGAL在肾小管上皮细胞缺氧/复氧过程中发挥的作用。方法构建高表达NGAL的载体pcDNA3.1(-)/myc-His B-NGAL,并转染HK-2细胞。将HK-2细胞分为未转染组和转染组,每组再分为缺氧/复氧处理组和未处理组。用western blot法检测转染后NGAL蛋白表达水平和缺氧/复氧后细胞自噬微管相关蛋白轻链3-Ⅱ(LC3-Ⅱ)表达水平,并用CellTiter-Blue细胞活性检测系统检测细胞活力。结果成功构建高表达NGAL的载体pcDNA3.1(-)/myc-His B-NGAL,转染HK-2细胞后,细胞NGAL蛋白表达水平高于未转染组(t=8.959,P<0.05)。缺氧1 h/复氧24 h后,转染组细胞LC3-Ⅱ表达水平高于未转染组(t=21.721,P<0.05),而细胞活力低于未转染组(t=-6.238,P<0.05)。结论在HK-2细胞缺氧/复氧状态下,NGAL能增强细胞自噬水平,导致细胞损伤加重甚至死亡。Objective To observe the effects of neutrophil gelatinase-associated lipocalin （NGAL） on the autophagy of kidney tubules epithelial cell line HK-2 during hypoxia/reoxygenation and explore the role of NGAL. Methods The vector pcDNA3.1 （ -）/myc-His B-NGAL was constructed for high expression of NGAL protein and transfected into HK-2 ceils. The cells were divided into the two groups, transfection and non-transfection, and each group included the subgroups of hypoxia/reoxygenation and untreated subgroup as controls. The levels of NGAL protein and microtubule-associated protein light chain 3-II （LC3-II） in autophagy following hypoxia/reox- ygenation were detected by western blot. The viability of HK-2 cells was measured by CellTiter-BlueTM Cell Viability Assay. Results The vector pcDNA3. 1 （-）/myc-His B-NGAL for high expression of NGAL was successfully constructed. The level of expressed NGAL protein in the group of transfected HK-2 ceils was higher than that in the group of non-transfected cells （t = 8. 959 ,P 〈 0.05 ）. The level of LC3-II in the transfected ceils after 1 hour of hypoxia followed by 24 hour of reoxygenation was higher than that in the non- transfected cells （ t = 21. 721, P 〈 0.05 ）, but the cellular viability was lower than that of non-transfected cells with statistically significant differences （ t = - 6. 238, P 〈 0.05 ）. Conclusion Undergoing the hypoxia/reoxygenation, NGAL may significantly enhance the autophagy level of HK-2 cells leading to aggravated damage for the cells even death.

关 键 词：中性粒细胞明胶酶相关脂质运载蛋白 自噬 肾小管上皮细胞 HK一2细胞 缺氧 复氧 

分 类 号：R446[医药卫生—诊断学]