RESUMO
Although non-viral vectors are relatively safe, they have very low gene transfection efficiency, especially in pancreatic islet cells. To provide information on the use of non-viral vectors for transfecting genes into pancreatic islet cells, a comparative evaluation of non-viral options was performed. In vitro experiments were used to compare the transfection efficiency of three classes of non-viral vectors: Effectene, polyethylenimine (PEI, 25 kDa) and hemagglutinating virus of Japan-envelope (HVJ-E), into insulinoma cells (INS-1) and rat islets. Vascular endothelial growth factor (VEGF) gene with hypoxia-inducible RTP801 promoter was delivered into rat islets with Effectene and VEGF secretion under hypoxia was measured in the culture media. Luciferase activity and GFP assays indicated that Effectene exhibited the highest transfection efficiency, and HVJ-E was not suitable for transfection into pancreatic beta-cells. The cytotoxicity of Effectene was found to be similar to that of 25-kDa PEI by 7-amino actinomycin D (7-AAD) flow cytometry and acridine orange/propidium iodide (AO/PI) assays. When RTP801 promoter-VEGF plasmid was delivered to rat islets with Effectene, VEGF secretion increased specifically in islets under hypoxia. In conclusion, Effectene showed higher gene-delivery efficiency for pancreatic islets compared with other classes of non-viral delivery systems and is promising as a gene delivery agent for pretransplant ex vivo gene therapy of islets.
Assuntos
Hipóxia Celular/fisiologia , Vetores Genéticos/química , Transfecção/métodos , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Citometria de Fluxo , Ilhotas Pancreáticas , Lipídeos/química , Masculino , Polietilenoimina/química , Ratos , Ratos Sprague-Dawley , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Islet transplantation is a promising strategy for treatment of diabetes. However, islets are exposed to hypoxia in the process of isolation and transplantation and prone to apoptosis. Vascular endothelial growth factor (VEGF) gene transfer is one of the promising strategies to address this problem. However, VEGF expression in the cells under normoxia is undesirable since it may induce pathological angiogenesis. Therefore, VEGF expression should be regulated to avoid this problem. In this study, hypoxia-inducible VEGF gene was transferred to islets using a non-viral carrier. Rat islets were transfected with high molecular weight PEI (25 kDa, PEI25K), low molecular weight PEI (2 kDa, PEI2K), and polyamidoamine dendrimer (PAMAM). PEI25K had higher transfection efficiency to rat islets than PAMAM or PEI2K. The hypoxia-inducible gene expression vector, pRTP801-Luc or pRTP801-VEGF was transferred to rat islets using PEI25K. Transfection assay with pRTP801-Luc showed that luciferase expression was induced in rat islets under hypoxia. In addition, transfer of pRTP801-VEGF showed that VEGF gene expression was higher under hypoxia than normoxia in rat islets. In conclusion, delivery of pRTP801-VEGF using PEI25K induces VEGF level specifically under hypoxia and may be useful for the development of anti-apoptotic strategies for islet transplantation.
Assuntos
Transplante das Ilhotas Pancreáticas/patologia , Polietilenoimina/química , Transfecção , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Técnicas de Cultura de Células , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dendrímeros , Ensaio de Imunoadsorção Enzimática , Humanos , Hipóxia/metabolismo , Luciferases/genética , Luciferases/metabolismo , Masculino , Poliaminas/química , Poliaminas/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Suínos , Fatores de TranscriçãoRESUMO
In this study, we developed an expression system of exendin-4, a glucagon-like peptide (GLP-1) analog, using a secretion signal peptide (SP) to facilitate exendin-4 secretion. For delivery of the exendin-4 expression system, high-molecular-weight polyethylenimine (25 kDa, PEI25k), low-molecular-weight polyethylenimine (2 kDa, PEI2k), and polyamidoamine (PAMAM) dendrimers were evaluated as gene carriers to Ins-1 beta cells. As a result, PEI25k showed the highest transfection efficiency. For the construction of the exendin-4 expression vector, DNA coding the SP sequence was inserted upstream of the exendin-4 cDNA, resulting in the construction of pbeta-SP-Ex-4. Transfection assay showed that the secretion level of exendin-4 increased in the pbeta-SP-Ex-4 transfected cells, compared with the pbeta-Ex-4 transfected cells. To identify the beta-cell protection effect of pbeta-SP-Ex-4 delivery, the Ins-1 beta cells were transfected with pbeta-SP-Ex-4 or pbeta-Ex-4 and incubated under normoxia or hypoxia. An MTT assay showed that the pbeta-SP-Ex-4 transfected cells had higher beta-cell viability than the pbeta-Ex-4 transfected cells under hypoxia. In addition, the pbeta-SP-Ex-4 transfected cells exhibited lower caspase-3 activity than the pbeta-Ex-4 transfected cells. Therefore, PEI25k/pbeta-SP-Ex-4 complex may be useful to protect isolated beta cells from apoptosis during transplantation.