腺病毒过表达TXNIP对INS-1胰岛细胞损伤和凋亡的影响  

Effect of adenovirus-mediated TXNIP overexpression on apoptosis and injury of INS-1 islet cell

作　　者：刘晓波[1] 杨啸[1] 姚艳玲[1] 张倩[1] 焦向英[1] 

机构地区：[1]山西医科大学生理学系,山西省太原市030001

出　　处：《中国心血管病研究》2014年第12期1127-1132,1151,1152,共8页Chinese Journal of Cardiovascular Research

基　　金：国家自然科学基金（项目编号：30800399）;山西省自然基金（项目编号：2014011049-12）;山西省高等学校创新人才支持计划;山西省回国留学人员科研资助项目（项目编号：2009-46）

摘　　要：目的 观察TXNIP过表达是否可以引起正常培养条件下的胰岛细胞发生损伤和凋亡,分析TXNIP引起细胞损伤和凋亡的下游途径,以进一步明确TXNIP在糖尿病中引起各器官细胞损伤的机制.方法 将使用正常糖脂浓度培养的胰岛细胞分为4组：正常培养组、Ad-eGFP空病毒组、Ad-TXNIP过表达组和Ad-TXNIPC247S点突变过表达组.结果 与Ad-eGFP空病毒组相比,两个过表达组的TXNIP mRNA水平、TXNIP蛋白水平均显著升高,表明病毒转染成功,TXNIP成功过表达.与空病毒组相比,Ad-TXNIP过表达组TXNIP与Trx的结合量升高（1.67±0.08比1.06±0.05,P＜0.05）,而Trx活性显著降低（0.42±0.11比1.13±0.14,P＜0.01）;LDH活性[（498.8±55.62）U/L比（266.2±36.49）U/L,P＜0.01]、caspase-3活性[（3.799±0.330）nmol·h-1·mg-1 pro比（0.979±0.100）nmol·h-1·mg-1 pro,P＜0.01]及p38激酶活性（2.45±0.22比1.10±0.08,P＜0.01）均显著增高.与Ad-TXNIP过表达组相比,C247S点突变的TXNIP过表达组TXNIP与Trx的结合量减少,Trx活性明显升高,LDH活性[（421.6±41.31）U/L比（498.8±55.62）U/L,P＜0.05]和caspase-3活性[（3.377±0.290）nmol·h-1·mg-1 pro比（3.799±0.330）nmol·h-1·mg-1 pro,P＜0.01]较低,p38激酶活性（0.80±0.07比2.45±0.22,P＞0.05）明显降低.结论 单纯过表达TXNIP可以引起正常糖脂浓度培养条件下的胰岛细胞发生损伤和凋亡.TXNIP介导细胞损伤和凋亡的机制一方面与其抑制Trx活性、增加自由基损伤、增加ASK1-p38激酶依赖的凋亡途径有关,同时也有不依赖结合抑制Trx的途径存在.TXNIP的表达上调是糖尿病引起胰岛细胞损伤和凋亡的重要原因.Objective To observe if TXNIP overexpression alone can cause islet cell apoptosis,Adenovirus carrying TXNIP gene was used to transfect INS-1 islet cells cultured in normal glucose and normal lipid concentration conditions.Meanwhile,adenovirus carrying a mutant C247S TXNIP gene,which lost its ability to attach and inhibit Trx by changing the cysteine at site 247 of TXNIP to serine,was also used to elucidate if TXNIP has additional effect on cell apoptosis independently of Trx.Methods INS-1 islet cells cultured in normal sugar conditions were randomly divided into four groups：normal cultured group,empty adenovirus vector group （AdeGFP）,TXNIP overexpression group（Ad-TXNIP-eGFP） and C247S mutated TXNIP overexpression group.Results Compared with empty adenovirus vector group,TXNIP mRNA and protein expression level,the combination of Trx and TXNIP,LDH activity,caspase-3 activity and p38 kinase activity in TXNIP overexpression group were significantly increased,while the Trx activity decreased significantly.In C247S mutated TXNIP overexpression group,parameters such as TXNIP mRNA and protein expression level,LDH activity,caspase-3 activity were also significantly increased,however,the combination of Trx and TXNIP,Trx activity,p38 kinase activity were not changed.Compared with TXNIP overexpression group,C247S mutated TXNIP overexpression group showed increased Trx activity and decreased combination of Trx and TXNIP and p38 kinase activity.Conclusion These results suggested that TXNIP overexpression can induce severe apoptosis and injury in islet cell even they are cultured in normal glucose and normal lipid concentration conditions.The mechanism involved is that TXNIP can bind and inhibit Trx,thus impaired its antioxidative and antiapoptotic function,and then enhanced free radical induced injury and p38 kinase dependent cell apoptosis.Trx independent pathway may also participated since C247S mutated TXNIP cannot bind to and inhibit Trx and have no effect on p38 kinase activity induced cell apoptosis.

关 键 词：硫氧还蛋白 硫氧还蛋白相互作用蛋白 胰岛细胞 凋亡 突变 

分 类 号：R587.1[医药卫生—内分泌]