摘要
目的:利用含绿色荧光蛋白的重组表达质粒pEG-FP-NeuroD做基因转染,观察其在肝癌细胞(HepG2)中的表达,探讨其在体外诱导肝细胞分泌胰岛素的可能性.方法:将酶切验证正确的重组质粒pEGFP-NeuroD,用脂质体法转染3种不同葡萄糖浓度培养的HepG2细胞,荧光显微镜下观测各组绿色荧光蛋白的表达情况;采用RT-PCR方法检测HepG2细胞中NeuroD-1及insulin mRNA的表达;采用Western Blot方法检测EGFP-NeuroD融合蛋白的表达.结果:①重组质粒体外成功转染入HepG2细胞,在荧光显微镜下可见强绿色荧光蛋白的表达,重组质粒转染率在30%～40%之间;②RT-PCR方法及Western Blot在3种不同葡萄糖浓度培养的细胞组中均检测到NeuroD-EGFP融合蛋白的表达,其中RT-PCR方法扩增出NeuroD-1目的片段大小为634 bp,而融合蛋白Mr大小约为67×103,但是3组间蛋白表达量均无明显差异;③RT-PCR法未检测到肝癌细胞胰岛素的分泌.结论:重组表达质粒pEGFP-NeuroD可体外转染入HepG2细胞,功能探讨提示单一NeuroD-1表达质粒的转染可能不足以诱导肝癌细胞分泌胰岛素.
AIM： To make gene transfection with pEGFP- NeurnD expression plasmid containing enhanced green fluorescent protein, to observe its expression in HepG2 cells and to detect whether it could induce hepatocytes to secrete insulin in vitro. METHODS： After identification, the recombinant plasmid pEGFP-NeuroD was transfected into HepG2 cells cultivated in three different concentrations of glucose culture solution by tech- niques of lipofectamine transfection. The green fluorescent protein expression was observed by fluorescence microscopy in the three groups and NeuroD-1 and insulin mRNA expression was detected by RT-PCR. Western Blot analysis was used to determine the expression of fusion protein NeuroD-EGFP. RESULTS： （1） The recombinant plasmid pEGFP-NeuroD was successfully transfected into HepG2 cells. The strong expression of green fluorescent pro- tein was observed by fluorescent microscopy and the transfection efficiency ranged from 30% to 40%. （2） The expression of NeuroD-EGFP was detected by RT-PCR and Western blot in three groups. 634 bp NeuroD-1 DNA fragment was amplified by RT-PCR and the molecular weight of fusion protein NeuroD-EGFP was 67 - 103. But there was no significant difference in the protein expression among the three groups. （3） No evidence of insulin secretion of hepatoma carcinoma cells was shown by RT-PCR. CONCLUSION ： Recombinant plasmid pEGFP- NeuroD is effectively expressed after being transfected into HepG2 ceils in vitro and it suggests that the transfection only with NeuroD-1 expression plasmid is not enough to induce hepatoma carcinoma ceils to secrete insulin.
出处
《第四军医大学学报》
北大核心
2009年第5期408-411,共4页
Journal of the Fourth Military Medical University
基金
重庆市自然科学基金(CSTC2007BB5281)
