腺病毒载体转染人HO-1基因增强成人胰岛细胞抗凋亡和胰岛素释放功能的研究

Transduction of heme oxygenase-1 gene to human Islet cells via adenovirus vector enhances anti-apoptosis function and insulin release

目的了解腺病毒载体转染人HO-1基因对体外培养的成人胰岛的作用,探索基因治疗在胰岛移植中的潜在应用价值。方法将成人胰岛分离纯化后分为3组:转染人HO-1基因组(Ad-HO-1组)、转染EGFP基因组(Ad-EGFP组)及对照组,采用携带人HO-1基因及EGFP基因的腺病毒作为载体对体外培养的成人胰岛进行转染,通过形态学观察、胰岛素释放试验及肿瘤坏死因子(TNF)α及放线菌酮诱导48 h后流式细胞仪检测凋亡。结果Ad-HO-1组的胰岛在高糖刺激下胰岛素分泌量为270 m IU/L±89 m IU/L,高于对照组(182 m IU/L±59 m IU/L)和Ad-EGFP组(189 m IU/L±88 m IU/L,P<0.05);诱导后Ad-HO-1组凋亡细胞的发生率为63%±11%,对照组为91%±11%,两组比较,P<0.01。结论使用腺病毒作为载体对体外培养的成人胰岛转染HO-1基因能够增加胰岛的抗凋亡能力、促进胰岛素的分泌。

Objective To investigate the effects of heme oxygenase-1 （ HO-1 ） gene on human islets in vitro, and to explore the potential value of gene therapy in clinical islet transplantation. Methods Adenovirus vector carrying human HO-1 gene （Ad-HO-1） or EGPF （Ad-EGFP） were established respectively. Human cadaveric pancreases were isolated, purified, cultured, and divided into 3 groups to be transfacted with Ad-HO-1, Ad-EGFP or blank vector. Human tumor necrosis factor and cyclohexamide （CHX） were added into the culture fluid of the pancreatic lslets. 48 hours later the pancreatic lslets were digested into single cells. Flow cytometry was used to detect the apoptosis. Glucose of the concentration of 16.7mmol/L was added into the culture fluid of the 3 groups of islet ceils. After 1-bour co-incubation radioimmunochemestry was used to detect the level of insulin in the supornatant. Results After stimulation of glucose the insulin concentration in the supornatant of the Ad-HO-1 group was 270 mIU/L ± 89 mIU/L, significantly higher than those of the Ad-EGFP group （ 189 mIU/L ± 88 mIU/L） and control group （182 mIU/L ±59 mIU/L, both P 〈0.05 ）. The apoptotic ratio of the Ad-HO-1 group was 63.1% ± 10. 9%, significantly lower than that of the control group （90. 9% ± 11.3%, P 〈0.01 ） after treatment with TNFaand CHX. Conclusion Transfection of Ad-HO-1 into human islets improves anti-apoptotic function in cultured human islets and promotes insulin release of human pancreatic islets.