缺氧环境下基因CRY1表达与大鼠肝细胞BRL-3A凋亡的关系  

The relationship between gene CRY1 and apoptosis in liver cells BRL-3A of rats under hypoxia

作　　者：钱琤 张育瑆[2] 王勇[3] 包小平[3] 赵文霞[3] 宋剑[3] 

机构地区：[1]浙江省湖州市妇幼保健院,湖州313000 [2]上海第二军医大学附属长征医院 [3]上海市江湾医院

出　　处：《浙江创伤外科》2016年第6期1027-1031,共5页Zhejiang Journal of Traumatic Surgery

基　　金：上海市卫生局科研计划项目(2010055)

摘　　要：目的观察模拟缺氧条件下大鼠BRL-3A细胞中基因CRY1与细胞凋亡及NF-Kappa B凋亡信号通路的关系。方法通过氯化钴体外模拟缺氧环境,观察大鼠肝BRL-3A细胞CRY1蛋白表达;筛选、构建CRY1mRNA慢病毒干扰载体,对BRL-3A细胞CRY1mRNA表达进行敲减,利用Western Blot法对BRL-3A细胞中CRY1干扰效能进行观察,并观察缺氧条件下转录因子CRY1敲减后BRL-3A细胞凋亡、NF-Kappa B、Caspase3蛋白表达。结果梯度浓度氯化钴模拟缺氧条件下BRL-3A细胞中CRY1表达随氯化钴浓度增加,表达逐渐下调;成功构建CRY1mRNA干扰载体pLenti6.3-EGFP-rCRY1-miR,有效敲减BRL-3A细胞中CRY1mRNA和蛋白表达,在100μM氯化钴模拟缺氧条件下YY1敲减后NF-Kappa B、Caspase3蛋白表达增加,凋亡持续增加。结论缺氧致大鼠BRL-3A细胞中转录因子CRY1表达下调,并通过NF-KB凋亡信号通过激活诱导细胞凋亡。Objective To evaluate the role of CRY1 in the apoptosis of BRL-3A cells under hypoxia and it ＇s relationship with NF-Kappa B apoptosis pathway. Methods The expression of CRY1 was detected in the liver cells BRL-3A of rats under gradient concentration（0-500 nM）of CoCl_2 on protein level. The miRNA targeting on CRY1 was applied by p Lenti6.3-EGFP-CRY1-miRNA vector,differential expression of CRY1 was detected between normoxic condition and mimic hypoxia by 100mmol/L CoCl_2 in BRL-3A cells by q RT-PCR,and western blot assay.p Lenti6.3-EGFP-YY1-miRNA vector on the apoptosis and the expression of NF-Kappa B,Caspase3 protein in BRL-3Acells under normoxic condition and mimic hypoxia were evaluated by Annexin V-PI staining and western blot assay. Results The decreased expression of CRY1 protein was observed in BRL-3A cells under hypoxia by gradient CoCl_2. The aberrant expression of CRY1 by miRNA vector upregualted the expression of NF-Kappa B,Caspase 3 protein and exaggerated the apoptosis in BRL-3A cells under hypoxia by 100mmol/L CoCl_2 when compared with normoxic condition. Conclusion Decreased expression of YY1 under mimic hypoxia induced the apoptosis in BRL-3A cells of rats through NF-Kappa B apoptosis pathway.

关 键 词：缺氧 CRY1 肝细胞 RNA干扰 凋亡 

分 类 号：R605.9[医药卫生—外科学]