人胰岛淀粉样多肽沉淀细胞模型的体外构建

Construction of human islet amyloid polypeptide expression vector

目的构建人胰岛淀粉样多肽(hIAPP)基因真核表达载体,并在无内源性IAPP表达的中国仓鼠卵巢(CHO)细胞株上进行体外表达实验,建立可产生hIAPP沉淀的细胞模型。方法应用RT-PCR技术扩增得到hIAPP cDNA,将其克隆至真核表达质粒pcDNA3.1(+)中,构建重组真核表达质粒pcDNA3.1(+)-IAPP;随后用lipofectamine 2000介导转染CHO细胞,以RT-PCR法检测重组质粒在mRNA水平的表达,以Thiaflavin S染色的方法检测重组质粒在蛋白水平的表达。结果构建的pcDNA 3.1-hIAPP真核表达体系成功转染体外培养的CHO细胞,目的基因在mRNA水平和蛋白水平均有表达,转染后培养48 h内的CHO细胞用Thioflavin S染色,与阴性对照相比较,证实有hIAPP的表达而导致细胞内淀粉样沉积发生。结论构建了hIAPP真核表达质粒,并成功在体外表达,从而建立了可产生IAPP淀粉样沉淀的体外哺乳动物细胞模型,为进一步研究胰岛淀粉样沉积的发病机制提供了良好的平台。

Objective To construct an eukaryotic expression vector of human islet amyloid polypeptide （hIAPP） , and to detect its expression in the cultured Chinese hamster ovary （CHO） cells with no endogenous IAPP, so as to establish a cell model which can form amyloid. Methods hIAPP cDNA was amplified by RT-PCR technique and was inserted into eukaryotic expression vector pcDNA3.1 （ ＋ ） to construct the recombinant expression plasmid pcDNA3.1 （ ＋ ） - 1APP. The recombinant plasmid was transfected into CHO cells by lipofectamine 2000. The transcription and the expression of IAPP in CHO cells were tested by RT-PCR and Thiaflavin S staining. Results The cultured CHO cells were successfully transfected with pcDNA3.1-hIAPP in vitro. The transcription of lAPP gene and expression of IAPP in transfected CHO cells were observed by RT-PCR and Thiaflavin S staining. Conclusion hlAPP expression vector is successfully constructed. A cell model which will form amyloid is established to provide a favorable platform for studying pathogenesis of islet amyloid.