【摘要】： 目的了解腺病毒載體轉(zhuǎn)染鼠白細(xì)胞介素10(IL-10)基因?qū)w外培養(yǎng)的胰島細(xì)胞的作用,探索基因治療在胰島移植中的潛在應(yīng)用價(jià)值。 方法自鼠脾細(xì)胞克隆IL-10CDNA,構(gòu)建攜帶有IL-10基因的重組腺病毒載體并感染RINm5F細(xì)胞,ELISA檢測IL-10基因表達(dá),檢測培養(yǎng)上清中NO水平,胰島素釋放實(shí)驗(yàn)進(jìn)行功能測定,流式細(xì)胞檢測細(xì)胞凋廣。 結(jié)果經(jīng)限制性內(nèi)切酶檢測和GFP表達(dá)證實(shí)成功地構(gòu)建了攜帶有IL-10基因的重組腺病毒載體并制備出高滴度重組病毒,病毒滴度為5.5×10~8pfu/ml。IL-10蛋白有效表達(dá),并具有抑制STZ誘導(dǎo)胰島β細(xì)胞凋亡、保擴(kuò)細(xì)胞胰島素分泌的功能。 結(jié)論成功地構(gòu)建了攜帶IL-10基因的重組腺病毒載休,并能有效表表達(dá),可增強(qiáng)胰島細(xì)胞的抗凋亡能力為進(jìn)一步研究IL-10基因臨床應(yīng)用奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R587.1;R346

【引證文獻(xiàn)】

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本文編號：2188425