真核过表达载体共转染3个胰岛发育关键基因在小鼠胎肝细胞内的表达

Expression of three key pancreatic islet development genes in mice fetal hepatocytes following co-transfection of eukaryotic expression vectors

背景:大量文献表明,向肝脏细胞引入一个多个胰岛发育的关键因子(如Pdx1、MafA及Ngn3)可以使肝细胞向胰岛细胞的方向进行转化。目的:构建以pcDNA3.1(+)为骨架的质粒载体并共同转染胰十二指肠同源框蛋白(Pdx1)、神经源素3(Ngn3)及V型肌腱膜纤维肉瘤癌基因同源基因A(MafA)至小鼠胎肝细胞并检测其表达情况。方法:以基因组或小鼠胰腺cDNA为模板扩增Pdx1、MafA及Ngn3三个目的基因,应用分子生物学技术,将3个目的片段克隆至真核表达载体pcDNA3.1(+)的多克隆位点中,构建小鼠系列真核过表达载体pcDNA3.1(+)-MafAORF,pcDNA3.1(+)-Pdx-1ORF,pcDNA3.1(+)-Ngn3ORF通过Lipofectamine2000介导的脂质体转染小鼠胎肝细胞系BNLCL.2,用反转录PCR以及间接免疫荧光法检测3个目的基因的表达情况。结果与结论:目的基因克隆正确,反转录PCR,间接免疫荧光法证实了脂质体转染后小鼠胎肝细胞中有Pdx1、Ngn3以及MafA的表达。结果表明,真核过表达载体pcDNA3.1(+)-MafAORF,pcDNA3.1(+)-Pdx-1ORF,pcDNA3.1(+)-Ngn3ORF可成功构建,并能够将Pdx1、Ngn3以及MafA三个基因转至小鼠胎肝细胞进行表达。

BACKGROUND：We have been studied that hepatocytes can be induced to differentiate into pancreatic B-cells through the introduction of a few factors of key islet development genes,such as pancreatic and duodenal homeobox-1（Pdx-1）,neurogenin 3（Ngn3）,v-maf musculoaponeurotic fibrosarcoma oncogene homolog A（MafA）.OBJECTIVE：To construct the plasmids using the pcDNA3.1（＋） frame to introduce the Pdx-1,MafA and Ngn3 into the mice fetal hepatocytes and to detect the expressions of these genes.METHODS：We amplified the target genes of Pdx-1,MafA and Ngn3 using templates of the cDNAs or the genome and then cloned to the eukaryotic expression vector pcDNA3.1（＋） multiple cloning sites.The eukaryotic overexpression vectors of pcDNA3.1（＋）-MafA ORF,pcDNA3.1（＋）-Pdx-1 ORF and pcDNA3.1（＋）-Ngn3 ORF were established by Lipofectamine2000 mediated liposome transfected with mouse fetal hepatocytes of BNL CL.2.The expressions of three target genes were tested through reverse-transcriptional polymerase chain reaction and indirect immunofluorescence.RESULTS AND CONCLUSION：The target genes were cloned correctly.The expressions of Pdx-1,Ngn3 and MafA in the fetal hepatocytes were validated by the detection of reverse-transcriptional polymerase chain and immunofluorescence test.We successfully constructed the eukaryotic overexpressed plasmids of pcDNA3.1（＋）-MafA ORF,pcDNA3.1（＋）-Pdx-1 ORF and pcDNA 3.1（＋）-Ngn3 ORF which could express the genes of Pdx-1,Ngn3 and MafA in the fetal hepatocytes.