使用腺病毒过表达TXNIP对H9C2心肌细胞损伤和凋亡的影响  

Effect of adenovirus-mediated TXNIP overexpression on apoptosis and injury of H9C2 cardiomyocytes

作　　者：姚艳玲[1] 杨啸[1] 薛晓维[1] 范丽芬[1] 焦向英[1] 

机构地区：[1]山西医科大学生理教研室,太原030001

出　　处：《生理学报》2013年第3期309-318,共10页Acta Physiologica Sinica

基　　金：supported by the National Natural Science Foundation of China(No.30800399);the Scientific and Technological Foundation for Distinguished Returned Overseas Chinese Scholars;Shanxi Province;China [No.2010(97)];the Scientific Research Foundation for Overseas Returnee of Shanxi Province;China(No.2009-46);Innovation Talents Support Program of Universities; Shanxi Province;China

摘　　要：本文旨在使用病毒转染技术,观察正常培养条件下的心肌细胞单纯过表达硫氧还蛋白相互作用蛋白(thioredoxin inter-acting protein,TXNIP)是否可以引起细胞损伤和凋亡的发生,并分析其作用途径。对数生长期的H9C2心肌细胞分为三组:正常培养组、空病毒(Ad-eGFP)组、TXNIP过表达(Ad-TXNIP-eGFP)组,使用正常糖脂浓度(5mmo1/L葡萄糖)的DMEM培养基,均于转染72h后收集细胞和培养基进行指标测定。结果显示,细胞转染成功,72h转染效率达到高峰。与Ad-eGFP组相比,Ad-TXNIP-eGFP转染组TXNIPmRNA(P<0.05)和蛋白表达(P<0.01)均明显升高;心肌caspase-3(P<0.05)和LDH活性明显升高(P<0.01);使用流式细胞仪测得细胞凋亡明显增加(P<0.01)。Trx活性、与Trx相关的自由基损伤以及介导氧化应激损伤凋亡途径的p38激酶的活性检测显示,与Ad-eGFP组相比,TXNIP过表达组的Trx活性明显降低(P<0.01),反映膜脂质过氧化损伤的指标丙二醛(malondial dehyde,MDA)、3-硝基酪氨酸明显升高(P<0.01),p38激酶活性明显升高(P<0.01)。这些结果提示,通过腺病毒转染单纯过表达TXNIP可以引起正常糖脂浓度培养条件下的心肌细胞发生损伤和凋亡,其具体机制与抑制Trx活性、增加自由基损伤和p38激酶介导的凋亡有关。Adenovirus transfection technique was used in the current study to show if thioredoxin-interacting protein （TXNIP） overexpression can induce cell apoptosis and injury in H9C2 cardiomyocytes cultured in normal glucose condition. And the mechanisms were then investigated. Briefly, H9C2 cardiomyocytes in logarithmic growth phase were randomly divided into three groups： normal cultured group, empty adenovirus vector group （Ad-eGFP） and TXNIP overexpression group （Ad-TXNIP-eGFP）. All cells were cultured in DMEM containing normal concentration of glucose （5 mmol/L） and lipid. 72 h after adenovirus transfection, cells and culture mediums were collected for further assay. The results showed that Ad-eGFP and Ad-TXNIP-eGFP adenovirus transfected H9C2 cells successfully, and the transfection efficiency reached the peak at 72 h. Compared with Ad-eGFP group, Ad-TXNIP-eGFP transfection significantly increased TXNIP mRNA （P 〈 0.05） and protein expression level （P 〈 0.01）. TXNIP overexpression induced remarkable cell apoptosis and injury as evidenced by increased caspase-3 activity （P 〈 0.05）, apoptotic rate （P 〈 0.01） and LDH activity （P 〈 0.01）. To further analysis the mechanisms of TXNIP-induced cell apoptosis, we also determined Trx activity, Trx related free radical injury and p38 kinase activation, which are involved in free radical induced apoptosis. The results showed that, compared with those in Ad-eGFP group, Trx activity was significantly decreased （P 〈 0.01）, while malondialdehyde （MDA）, 3-nitrotyrosine contents and p38 kinase activity were significantly increased （P 〈 0.01） in TXNIP overexpression group. These results suggest that TXNIP over-expression alone can induce severe apoptosis and injury in H9C2 cardiomyocytes even they are cultured in normal glucose and lipid concentration conditions. The mechanism involved is that overexpressed TXNIP can bind and inhibit Trx, impairs its antioxidative and antiapoptotic function, and then increases free radica

关 键 词：硫氧还蛋白 硫氧还蛋白相互作用蛋白 心肌细胞 凋亡 

分 类 号：R541[医药卫生—心血管疾病]