CAJ | 학술논문

目的研究A20基因、血红素加氧酶1基因(heme oxygenase 1 gene,HO-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-α(TNF-α)诱导的胰岛细胞凋亡作用的影响.方法构建A20、HO-1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-I蛋白表达.超敏ELISA试剂盒检测胰岛素浓度.胰岛经CHX+ TNF-α处理48 h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-3的活化.结果 (1)分别以A20和HO-1慢病毒转染胰岛检测表明A20和HO-1蛋白均呈高表达.(2)胰岛细胞经培养48h和96 h,转基因各组胰岛素浓度高于空白对照组(P＜0.01).(3)CHX+ TNF-α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HO-1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) μg/ml]和空白对照组[(8.86±0.38) μg/ml,P＜0.001].Western印迹法检测显示联合转A20/HO-1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-1蛋白具有抗CHX+ TNF-α诱导的胰岛凋亡作用,联合转A20和HO-1基因有协同抗凋亡效应.
목적 연구A20기인、혈홍소가양매1기인(heme oxygenase 1 gene,HO-1)전염재대서이도세포중적표체정황급대체외배양조건하이도활성、길항방선균동(CHX)화종류배사인자-α(TNF-α)유도적이도세포조망작용적영향.방법 구건A20、HO-1화증강록색형광단백(EGFP)만병독전이재체,형광현미경련속관찰EGFP표체정황,평고전기인효솔、학정유도조망시궤.Western인적측정A20화HO-I단백표체.초민ELISA시제합검측이도소농도.이도경CHX+ TNF-α처리48 h진행TUNEL、류식세포의검측조망솔.Western인적검측반광안산단백매-3적활화.결과 (1)분별이A20화HO-1만병독전염이도검측표명A20화HO-1단백균정고표체.(2)이도세포경배양48h화96 h,전기인각조이도소농도고우공백대조조(P＜0.01).(3)CHX+ TNF-α유도이도세포조망후,전A20조이도소농도위(93.58 ±4.12) μg/ml、전HO-1조이도소농도위(88.98±4.77) μg/ml,연합전A20화HO-1조이도소농도(103.33 ±3.16) μg/ml,균고우전EGFP조[(9.03±0.65) μg/ml]화공백대조조[(8.86±0.38) μg/ml,P＜0.001].Western인적법검측현시연합전A20/HO-1기인조활화형반광안산단백매-3표체수평최저.결론 원대이도세포중고효표체적A20화HO-1단백구유항CHX+ TNF-α유도적이도조망작용,연합전A20화HO-1기인유협동항조망효응.
Objeetive To establish the method for cotransferring human A20 gene and human heme oxygenase-1 (HO-1) gene into the isolated rat islets using lentiviral transfection system,and to study the protective effect of A20 and HO-1 protein against the apoptosis induced by cycloheximide (CHX) and TNFα,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-α,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 fromA20 and/or HO-1 transgenic islets were significantly higher than that in non-transgenic controls (P ＜0.01).(3)Mter 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-1 co-transfected group [(103.33 ± 3.16) μg/ml] were significantly higher than that in the EGFP group [(9.03 ± 0.65) μg/ml] and the control group [(8.86 ± 0.38) μg/ml] (P ＜0.001).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-expressed A20 and HO-1 protein delivered via lentivirus could preserve rats 'islets function and act against the apoptosis induced by CHX and TNF-α.