应用RNA干扰技术检测ASCT2功能

Measuring the function of glutamine transporter ASCT2 by RNA interference

目的谷氨酰胺(glutamine,GLN)是肠道细胞的主要氧化燃料,必须通过载体的转运才能进入细胞内,其中载体ASCT2起重要作用,文中将应用RNA干扰(RNA interference,RNAi)技术研究肠黏膜上皮细胞株(intestinal epithelial cell,IEC)-6细胞膜GLN载体ASCT2的转运功能。方法体外培养IEC-6,RNAi慢病毒颗粒进行细胞感染,3 d后荧光显微镜下观察绿色荧光蛋白(green fluorescent protein,GFP)表达情况,5 d后采用RT-PCR检测载体ASCT2的mRNA表达,7 d后采用Western blot检测其蛋白表达,同时测定[3 H]-L-GLN摄取率。结果病毒转染细胞中均有GFP表达,载体ASCT2的mRNA表达下降60%,蛋白表达几近消失,[3H]-L-GLN摄取率下降15%。结论通过慢病毒载体介导的RNAi这一方便有效的方法,证实了ASCT2载体在整个细胞的GLN转运中起15%以上的作用。

] Objective Glutamine,as the main oxidative fuel of enterocytes,enters the enterocyte primarily via amino acid transporters,in which AST2 plays an important role.The aim of this study was to investigate the function of the glutamine transporter ASCT2 in intestinal epithelial cell line 6(IEC-6) by RNA interference(RNAi).Methods Rat IEC-6 was incubated in vitro and transfected with the siRNA mediated by lentiviral vectors.The expression of green fluorescent protein(GFP) was detected by fluorescent microscope on the 3rd day of transfection;the mRNA expression of ASCT2 estimated by RT-PCR on the 5th day the protein expression of ASCT2 determined by Western-blot,and meanwhile the -L-glutamine uptake measured on the 7th day.Results GFP was found in all the transfected cells.The mRNA expression of ASCT2 was decreased by 60% and its protein expression was hardly observed.The total uptake of -L-glutamine was reduced by more than 15%.Conclusion RNAi mediated by lentivirus-based vectors is a convenient and effective method to test the function of ASCT2,which may contribute over 15% of total glutamine uptake.