鼠白细胞介素10重组腺病毒在RINM5F细胞的表达及生物活性
发布时间：2018-08-17 17:53
【摘要】： 目的了解腺病毒载体转染鼠白细胞介素10(IL-10)基因对体外培养的胰岛细胞的作用,探索基因治疗在胰岛移植中的潜在应用价值。 方法自鼠脾细胞克隆IL-10CDNA,构建携带有IL-10基因的重组腺病毒载体并感染RINm5F细胞,ELISA检测IL-10基因表达,检测培养上清中NO水平,胰岛素释放实验进行功能测定,流式细胞检测细胞凋广。 结果经限制性内切酶检测和GFP表达证实成功地构建了携带有IL-10基因的重组腺病毒载体并制备出高滴度重组病毒,病毒滴度为5.5×10~8pfu/ml。IL-10蛋白有效表达,并具有抑制STZ诱导胰岛β细胞凋亡、保扩细胞胰岛素分泌的功能。 结论成功地构建了携带IL-10基因的重组腺病毒载休,并能有效表表达,可增强胰岛细胞的抗凋亡能力为进一步研究IL-10基因临床应用奠定了基础。
[Abstract]:Objective to investigate the effect of adenovirus vector transfection of mouse interleukin 10 (IL-10) gene on cultured islet cells in vitro and to explore the potential value of gene therapy in islet transplantation. Methods IL-10 CDNAwas cloned from mouse spleen cells. Recombinant adenovirus vector carrying IL-10 gene was constructed and IL-10 gene expression was detected by Elisa in infected RINm5F cells. No level in culture supernatant was detected. The function of insulin release assay was measured. Flow cytometry was used to detect the wide expression of IL-10 gene. Results the recombinant adenovirus vector carrying IL-10 gene was successfully constructed by restriction endonuclease detection and GFP expression, and the recombinant virus with high titer was prepared. The virus titer was 5.5 脳 10~8pfu/ml.IL-10 protein effectively expressed. It can inhibit the apoptosis of islet 尾 cells induced by STZ and protect the insulin secretion of expanded cells. Conclusion the recombinant adenovirus carrying IL-10 gene was successfully constructed and expressed effectively, which can enhance the anti-apoptosis ability of islet cells and lay a foundation for the further study of clinical application of IL-10 gene.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R587.1;R346
本文编号：2188425
[Abstract]:Objective to investigate the effect of adenovirus vector transfection of mouse interleukin 10 (IL-10) gene on cultured islet cells in vitro and to explore the potential value of gene therapy in islet transplantation. Methods IL-10 CDNAwas cloned from mouse spleen cells. Recombinant adenovirus vector carrying IL-10 gene was constructed and IL-10 gene expression was detected by Elisa in infected RINm5F cells. No level in culture supernatant was detected. The function of insulin release assay was measured. Flow cytometry was used to detect the wide expression of IL-10 gene. Results the recombinant adenovirus vector carrying IL-10 gene was successfully constructed by restriction endonuclease detection and GFP expression, and the recombinant virus with high titer was prepared. The virus titer was 5.5 脳 10~8pfu/ml.IL-10 protein effectively expressed. It can inhibit the apoptosis of islet 尾 cells induced by STZ and protect the insulin secretion of expanded cells. Conclusion the recombinant adenovirus carrying IL-10 gene was successfully constructed and expressed effectively, which can enhance the anti-apoptosis ability of islet cells and lay a foundation for the further study of clinical application of IL-10 gene.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R587.1;R346
【引证文献】
相关博士学位论文 前1条
1 徐爱晶;IGF-1和IL-10基因转导对NOD鼠1型糖尿病的防治作用及机制探讨[D];青岛大学;2011年
，本文编号：2188425
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