Objective To investigate in vitro heme oxygenase-1 gene (HO-1) delivery to human pancreatic islets by adenovirus vectors. Methods Recombinant adenovirus containing HO-1 or enhanced green fluorescent protein gene(EGFP) was generated by using the AdEasy System. The purified human pancreatic islets were infected with recombinant adenovirus vectors at various multiplicity of infection (MOI). Transduction was confirmed by fluorescence photographs and Western blot. Glucose-stimulated insulin secretion was detected by using Human insulin radioimmunoassay kits and was used to assess the function of human islets infected by recombinant adenovirus.Results Viral titers of Ad-hHO-1 and Ad-EGFP were 1.96×109 and 1.99×109 pfu/mL, respectively. Human pancreatic islets were efficiently infected by recombinant adenovirus vectors in vitro. Transfection of human islets at an MOI of 20 did not inhibit islet function. Recombinant adenovirus mediated HO-1gene transfer significantly improved the islet function of insulin release when simulated by high level glucose. Conclusion Recombinant adenovirus is efficient to deliver exogenous gene into human pancreatic islets in vitro. HO-1 gene transfection can improve human islet function.
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。
