低氧条件下B淋巴细胞瘤-2/腺病毒E1B19 000相互作用蛋白3对人真皮微血管内皮细胞迁移和运动性的影响及其机制  

Effects of B-cell lymphoma-2/adenovirus E1B 19000 interacting protein 3 on the migration and motility of human dermal microvascular endothelial cells under hypoxia and the mechanism

作　　者：张均辉 张琼 贾杰只 李红梅 张灿 胡炯宇 张东霞 黄跃生 Zhang Junhui;Zhang Qiong;Jia Jiezhi;Li Hongmei;Zhang Can;Hu Jiongyu;Zhang Dongxia;Huang Yuesheng(Department of Endocrinology,the First Affiliated Hospital of Army Medical University(the Third Military Medical University),Chongqing 400038,China;State Key Laboratory of Trauma,Burns and Combined Injury,Institute of Burn Research,the First Affiliated Hospital of Army Medical University(the Third Military Medical University),Chongqing 400038,China;Department of Oncology,the First Affiliated Hospital of Army Medical University(the Third Military Medical University),Chongqing 400038,China;Department of Plastic Surgery,the First Affiliated Hospital of Army Medical University(the Third Military Medical University),Chongqing 400038,China;Department of Wound Repair,Institute of Wound Repair,Shenzhen People′s Hospital,the First Affiliated Hospital of Southern University of Science and Technology,the Second Clinical Medical College of Jinan University,Shenzhen 518020,China)

机构地区：[1]陆军军医大学(第三军医大学)第一附属医院内分泌科,重庆400038 [2]陆军军医大学(第三军医大学)第一附属医院全军烧伤研究所,创伤、烧伤与复合伤国家重点实验室,重庆40003 [3]陆军军医大学(第三军医大学)第一附属医院肿瘤科,重庆400038 [4]陆军军医大学(第三军医大学)第一附属医院整形外科,重庆400038 [5]深圳市人民医院南方科技大学第一附属医院暨南大学第二临床医学院创面修复科,创面修复研究所518020

出　　处：《中华烧伤杂志》2021年第1期9-16,共8页Chinese Journal of Burns

基　　金：国家重点基础研究发展计划(2017YFC1103302)。

摘　　要：目的探讨低氧条件下B淋巴细胞瘤-2/腺病毒E1B 19000相互作用蛋白3(BNIP3)对人真皮微血管内皮细胞(HDMEC)迁移和运动性的影响及其机制。方法采用实验研究方法。(1)取HDMEC,采用随机数字表法(下同)分成行常规培养的常氧组及采用体积分数2%氧气低氧处理相应时间点的低氧6、12、24 h组,采用蛋白质印迹法检测细胞中BNIP3及微管相关蛋白1轻链3Ⅱ(LC3Ⅱ)的蛋白表达。(2)取HDMEC,分成常氧+空载组、常氧+BNIP3敲减组、低氧+空载组、低氧+BNIP3敲减组,分别转染空载病毒或BNIP3敲减病毒并进行常氧或低氧处理6 h,采用蛋白质印迹法和免疫荧光染色法检测BNIP3的蛋白表达;采用划痕试验检测划痕后24 h的划痕面积,并计算划痕愈合率;在活细胞工作站测算3 h内细胞运动的曲线距离,计算运动速度。(3)取HDMEC,同实验(2)分组及处理,采用蛋白质印迹法和免疫荧光染色法检测LC3Ⅱ的蛋白表达。以上实验样本数均为3。对数据行单因素方差分析及LSD检验。结果(1)与常氧组比较,低氧6、12、24 h组细胞BNIP3及LC3Ⅱ的蛋白表达量显著增加(P<0.01)。(2)培养6 h,与低氧+空载组比较,常氧+空载组和低氧+BNIP3敲减组细胞BNIP3的蛋白表达量显著下降(P<0.05或P<0.01)。常氧+空载组和常氧+BNIP3敲减组细胞中表示BNIP3蛋白表达的红色荧光较弱,低氧+空载组细胞红色荧光较强,低氧+BNIP3敲减组细胞中红色荧光较低氧+空载组明显减弱。划痕后24 h,低氧+空载组细胞划痕基本愈合,其他3组细胞剩余划痕面积较大。常氧+空载组、常氧+BNIP3敲减组、低氧+空载组、低氧+BNIP3敲减组细胞划痕愈合率分别为(61±4)%、(58±4)%、(88±4)%、(57±4)%。低氧+空载组细胞划痕愈合率明显高于常氧+空载组(P<0.01)和低氧+BNIP3敲减组(P<0.05)。观察3 h内,低氧+空载组细胞运动范围较常氧+空载组显著增大,低氧+BNIP3敲减组细胞运动范围较低氧+空载组明显�Objective To explore the effects of B-cell lymphoma-2/adenovirus E1B 19000 interacting protein 3(BNIP3)on the migration and motility of human dermal microvascular endothelial cells(HDMECs)under hypoxia and the mechanism.Methods The experimental research method was applied.(1)HDMECs were divided into normoxia group received routine culture and hypoxia 6,12,24 h groups treated under hypoxia with oxygen volume fraction of 2%for corresponding time according to the random number table(the same grouping method below).Western blotting was used to detect the protein expressions of BNIP3 and microtubule-associated protein 1 light chain 3Ⅱ(LC3Ⅱ)in HDMECs.(2)HDMECs were divided into normoxia+unloaded group,normoxia+BNIP3 knockdown group,hypoxia+unloaded group,and hypoxia+BNIP3 knockdown group which were transfected with unloaded virus or BNIP3 knockdown virus and were subjected to normoxic or hypoxic treatment.The BNIP3 protein expression was detected by Western blotting and immunofluorescence staining.The scratch area at 24 h post scratching was detected by scratch test,and the healing rate of scratch was calculated.The curve distance of cell movement was measured with the living cell workstation,and the speed of movement was calculated within 3 hours.(3)HDMECs were grouped and treated as experiment(2).Western blotting and immunofluorescence staining were performed to detect the protein expression of LC3Ⅱ.The number of sample was 3 in the above-mentioned experiments.Data were statistically analyzed with one-way analysis of variance and least significant difference test.Results(1)Compared with those of normoxia group,the protein expressions of BNIP3 and LC3Ⅱof cells in hypoxia 6,12,24 h groups were significantly increased(P<0.01).(2)After 6 hours of culture,compared with that of hypoxia+unloaded group,the BNIP3 protein expressions of cells in normoxia+unloaded group and hypoxia+BNIP3 knockdown group were significantly decreased(P<0.05 or P<0.01).The red fluorescence denoting BNIP3 protein expression of cells in norm

关 键 词：细胞低氧 细胞迁移分析 细胞运动 自噬 人真皮微血管内皮细胞 B淋巴细胞瘤-2/腺病毒E1B19000相互作用蛋白3 

分 类 号：R619+.6[医药卫生—外科学] R62[医药卫生—临床医学]