Enhanced protection of Ins-1 β cells from apoptosis under hypoxia by delivery of DNA encoding secretion signal peptide-linked exendin-4
Enhanced protection of Ins-1 β cells from apoptosis under hypoxia by delivery of DNA encoding secretion signal peptide-linked exendin-4
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DOI：
10.1080/10611860902718664
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发表时间：
2009-04-01
影响因子：
4.5
通讯作者：
Lee, Minhyung
中科院分区：
文献类型：
--
作者：
Kim, Hyun Ah;Lee, Suyeon;Lee, Minhyung
在这项研究中，我们建立了一种胰升糖素样肽(GLP-1)类似物Exendin-4的表达系统，利用分泌信号肽(SP)促进Exendin-4的分泌。为了将exendin-4表达系统传递给INS-1β细胞，评价了高分子量聚乙烯亚胺(25 kDa，PEI25k)、低分子聚乙烯亚胺(2 kDa，PEI2k)和聚酰胺胺(PamAM)树枝状大分子作为INS-1β细胞的基因载体。结果表明，PEI25k的转染率最高。为了构建exendin-4表达载体，将编码SP序列的DNA插入exendin-4基因的上游，构建了pβ-SP-Ex-4。转染实验表明，与pβ-Ex-4相比，pβ-SP-Ex-4转染组细胞的exendin-4分泌水平增加。为确定p-β-SP-Ex-4对胰岛β细胞的保护作用，将p-SP-Ex-4或pβ-Ex-4分别导入INS-1β细胞，在常氧或低氧条件下孵育。四甲基偶氮唑盐比色试验表明，p-β-SP-Ex-4转基因细胞在低氧条件下具有比pβ-Ex-4转基因细胞更高的β细胞活力。此外，pβ-SP-Ex-4转染组细胞的caspase-3活性低于pβ-Ex-4转染组。因此，PEI25k/pβ-SP-Ex-4复合体可能在移植过程中对分离的β细胞起到保护作用。
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 p beta-SP-Ex-4. Transfection assay showed that the secretion level of exendin-4 increased in the p beta-SP-Ex-4 transfected cells, compared with the p beta-Ex-4 transfected cells. To identify the beta-cell protection effect of p beta-SP-Ex-4 delivery, the Ins-1 beta cells were transfected with p beta-SP-Ex-4 or p beta-Ex-4 and incubated under normoxia or hypoxia. An MTT assay showed that the p beta-SP-Ex-4 transfected cells had higher beta-cell viability than the p beta-Ex-4 transfected cells under hypoxia. In addition, the p beta-SP-Ex-4 transfected cells exhibited lower caspase-3 activity than the p beta-Ex-4 transfected cells. Therefore, PEI25k/p beta-SP-Ex-4 complex may be useful to protect isolated beta cells from apoptosis during transplantation.