联合转染A20、HO—1基因对大鼠胰岛抗凋亡能力影响的研究  

Protective effect of cotransfection of A20 and HO-1 gene against the apoptosis induced by TNF-α in rat islets in vitro

作　　者：陆忠[1] 沈水仙[1] 支涤静[1] 徐虹[2] 郭礼和[3] 罗飞宏[3] 

机构地区：[1]复旦大学附属儿科医院内分泌遗传代谢科,上海201102 [2]复旦大学附属儿科医院肾内科,上海201102 [3]中国科学院上海生化细胞研究所

出　　处：《中华儿科杂志》2013年第6期420-425,共6页Chinese Journal of Pediatrics

基　　金：国家自然科学基金（30470808）

摘　　要：目的研究A20基因、血红素加氧酶1基因（heineoxygenase1gene，110-1）转染在大鼠胰岛细胞中的表达情况及对体外培养条件下胰岛活性、拮抗放线菌酮（CHX）和肿瘤坏死因子．d（TNF—d）诱导的胰岛细胞凋亡作用的影响。方法构建A20、HO—J和增强绿色荧光蛋白（EGFP）慢病毒转移载体，荧光显微镜连续观察EGFP表达情况，评估转基因效率、确定诱导凋亡时机。Western印迹测定A20和HO-J蛋白表达。超敏ELISA试剂盒检测胰岛素浓度。胰岛经CHX＋TNF．仪处理48h进行TUNEL、流式细胞仪检测凋亡率。Western印迹检测半胱氨酸蛋白酶一3的活化。结果（1）分别以A20和HO．，慢病毒转染胰岛检测表明A20和HO—J蛋白均呈高表达。（2）胰岛细胞经培养48h和96h，转基因各组胰岛素浓度高于空白对照组（P〈0．01）。（3）CHX＋TNF-0【诱导胰岛细胞凋亡后，转A20组胰岛素浓度为（93．58±4．12）μg/ml、转110-J组胰岛素浓度为（88．98±4．77）μg／ml，联合转A20和HO-1，组胰岛素浓度（103．33±3．16）μg／ml，均高于转EGFP组[（9．03±0．65）Ixg／m1]和窄白对照组[（8．86±0．38）肌∥ml，P〈0．001]。Western印迹法检测显示联合转A20／HO-J基因组活化型半胱氨酸蛋白酶-3表达水平最低。结论原代胰岛细胞中高效表达的A20和HO—j蛋白具有抗CHX＋TNF—d诱导的胰岛凋亡作用，联合转A20和HO—J基因有协同抗凋亡效应。Objective To establish the method for cotransferring human A20 gene and human heine oxygenase-1 （HO-1） gene into the isolated rat islets using lentiviral transfection system, and to study the protective effect of A20 and HO-I protein against the apoptosis induced by cycloheximide （CHX） and TNF- a, and finally to explore the underlying mechanism. Method The A20 gene and HO-1 gene were cloned and inserted into the lentiviral transfection system. The efficacy of gene transfer was measured by the intensity of the enhanced green fluorescent protein （EGFP） fluorescence-positive islets. Western blot was applied to verify the expression of the A20 and HO-1 genes. To induce apoptosis in vitro, the isolated islets were treated with CHX ＋ TNF-a, terminal deoxynucleotidyl transferase dUTP nick end labeling （TUNEL） and the fluorescence-activated cell sorting （FACS） methods were used to evaluate the apoptosis of the islet cells and Western blot was used to detect caspase-3 activation. Result （1） A20 and HO-1 genes were introduced into the isolated islets by lentiviral transfection, both of the genes were highly expressed in the islets after 96 hours culture detected by Western blot method. （2） The insulin levels in the cell culture medium from A20 and/or HO-1 transgenic islets were significantly higher than that in non-transgenic controls （P 〈 0. O1 ）. （3）After CHX ＋ TNF-alpha treatment, the cell culture medium insulin concentration in the A20 gene transfected group [ （93.58 ＋ 4. 12） μg/ml], HO-1 gene transfected group [ （88.98 ＋ 4. 77） μg/ml and A20/HO-I co-transfected group [ （ 103.33 ＋ 3. 16） μg/ml] were significantly higher than that in the EGFP group [ （9. 03 ＋ 0. 65） p,g/ml ] and the control group [ （8.86 ＋ 0. 38） μg/mll （P 〈O. 001 ）. Minimum expression level of the activated caspase-3 was found in the A20/HO-1 co-transfected group. Conclusion The lentiviral gene transfer system was an efficient and stable gene transfer vector, the over-ex

关 键 词：胰岛 血红素加氧酶1 基因 转染 凋亡 

分 类 号：R587[医药卫生—内分泌]