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目的:构建小鼠PPA1重组腺病毒,观察重组腺病毒在小鼠胰岛和β-TC6细胞中的表达,以及对脂肪酸引起的胰岛β细胞凋亡的影响。方法:将目的基因PPA1插入到腺病毒穿梭质粒pAdtrack-CMV中,质粒经PCR鉴定正确后用PmeⅠ酶切线性化,转到含有腺病毒骨架pAdeasy-1的BJ5183细菌中进行同源重组,重组成功的质粒经PacⅠ酶切线性化后转染QBI-293A细胞,经包装得到Ad-PPA1腺病毒。根据绿色荧光蛋白(GFP)检测病毒滴度和感染效率。用病毒感染小鼠胰岛和β-TC6细胞,以Western blot法检测PPA1蛋白表达水平。Hoechst染色法检测PPA1对细胞凋亡的影响。结果:重组腺病毒载体pAdeasyPPA1经酶切鉴定确认构建成功,将pAdeasy-PPA1转染QBI-293A细胞,观察细胞病变效应提示病毒成功包装。重组腺病毒能够有效感染小鼠胰岛和β-TC6细胞并成功过表达PPA1蛋白。Hoechst染色结果表明过表达PPA1可保护脂肪酸引起的胰岛β细胞凋亡。结论:成功构建携带小鼠PPA1基因的重组腺病毒,并证实过表达PPA1具有抗凋亡的作用,为进一步研究PPA1在胰岛β细胞中的功能奠定了基础。
OBJECTIVE: To construct mouse PPA1 recombinant adenovirus and observe the expression of recombinant adenovirus in mouse pancreatic islet and β-TC6 cells and its effect on fatty acid-induced pancreatic β-cell apoptosis. Methods: The target gene PPA1 was inserted into the adenovirus shuttle plasmid pAdtrack-CMV. The plasmid was identified by PCR and linearized with PmeI. The plasmid was transformed into BJ5183 containing adenovirus backbone pAdeasy-1 and recombined successfully The plasmids were linearized with Pac I and transfected into QBI-293A cells. The recombinant adenoviruses were packaged to obtain Ad-PPA1 adenovirus. Virus titers and infection efficiencies were determined based on green fluorescent protein (GFP). Mouse islets and β-TC6 cells were infected with virus and the expression of PPA1 protein was detected by Western blot. Hoechst staining was used to detect the effect of PPA1 on apoptosis. Results: The recombinant adenoviral vector pAdeasyPPA1 was identified by restriction enzyme digestion. The recombinant plasmid pAdeasy-PPA1 was transfected into QBI-293A cells. The cytopathic effect was observed and the virus was successfully packaged. The recombinant adenovirus can effectively infect mouse islets and β-TC6 cells and overexpress PPA1 protein. Hoechst staining showed that overexpression of PPA1 protected fatty acid-induced pancreatic β-cell apoptosis. Conclusion: The recombinant adenovirus carrying mouse PPA1 gene was successfully constructed and confirmed that overexpression of PPA1 has the anti-apoptotic effect, which laid the foundation for further study on the function of PPA1 in pancreatic β-cell.