Reprogramming Of Mice Fetal Hepatocytes Into Islet-like Cells By The Nucleofection With Polycistronic Vectors

[Background]The prevalence of diabetes rockets nowadays in our country along with the fast economic developing,the survey held by CDS in2008revealed that there were already11percent of the adult Chinese population who had got diabetes, not to mention the rate of15percent on the IGT(Impaired Glucose Tolerance) situation. The chronic complications of DM are the most threatening things to the patients, which also has become great burden of the whole country. The central part of the pathogenesis is prevalently recognized as the the insulin-deficiency caused by the dysfunction or failure of islet B cells which represents the damage of B cell. A significant method to prevent and treat DM is to maintain and make up the loss of the islet cell mass. The Edmonton Protocol apparently brought a ray of hope to the islet transplat, but was restricted by the extreme deficiency of donor cells and the immunological rejection, therefore, the source of the safe and abundant insulin-secreting cells has become one of the utmost problem of islet transplant which makes the islet neogenesis the focus of the cell-replacement therapy for DM. Although the transformation or transdifferentiation of the stem cells and somatic cells into insulin-secreting cells seems definitely to be the hot point of the cell-replacement therapy, the complicated procedure, the low transformation rate and biological function all make those protocols hard to carry out, not to mention the tumorgenesis, virus infection, immunological reaction and the systematic toxicity. Recently, the researchers have established another way of cell-reprogramming to transdifferentiate hepatocytes into insulin-secreting cells, the results demonstrated higher expression of insulin than the induction with regeneration factors and poly-peptide, along with the fluent steps, the direct somatic-somatic transdifferentiation, and the rich cell source. The pancreas and the liver share the same endodermal origin in the development of human body. It is the theoretical basis of the liver to pancreatic B-cell transdifferentiation. We have been studying that hepatocytes can be induced into pancreatic B-cells through the introduction of a few factors on islet development (such as Pdx-1,MafA and Ngn3). In the meantime, the multiple-cistronic plasmid vectors could provide a simple platform for the co-expression of a few transcription factors with no repeated clonal construction, no tumorgenesis activation and toxicity. Recent researches have already proved the feasibility of the of Pdx-1and Ngn3co-expression, therefore, we determined to administrate the procedure in Co-expression of Pdx-1,Ngn3and MafA through multi-cistronic vector in hepatocytes to activate the transcription of various islet-related genes,even to create the insulin secretion, hoping to solve the problems we mentioned above.We first built the plasmids of pcDNA3.1(+)-mafa ORF+ngn33’UTR+pdx13’UTR and pcDNA3.1(+)-ngn3ORF+pdx13’UTR, then utilized the lipofection to introduce those factors into mice fetal hepatocytes. Detections have been made to confirm the ectopic expression of Pdx-1, Ngn3and MafA. [Objective]1. To construct the plasmid vectors that could express Ngn3,Pdx1and MafA.2. To Observe the expression both on transcriptional and translational level of these three target genes and the islet-related gene.[Methods]1. We amplified the target genes of Pdx-1,MafA and Ngn3using templates of the cDNAs or the genome and generated the plasmids of pcDNA3.1(+)-mafa ORF+ngn33’UTR+pdx13’UTR and pcDNA3.1(+)-ngn3ORF+pdx13’UTR,2. We transfected the BNL CL.2cells (stable mice fetal liver cell line) with the plasmids mentioned above3. We detected the expressions of the three target genes (Pdx-1, Ngn3, MafA) and a islet-related gene Ins2through Reverse-Transcriptional PCR and the encoded protein of Pdx-1,Ngn3,MafA and Insulin through indirect Immunofluorescence test.[Results]1. Sequencing results shows that the target genes are cloned correctly which means we have successfully construct the plasmids of pcDNA3.1(+)-mafa ORF+ngn33’UTR+pdx13’UTR and pcDNA3.1(+)-ngn3ORF+pdx13’UTR.2. After the transfection and normal cell culture, the expressions of Pdx-1, Ngn3and MafA and Ins2in the BNL CL.2cells are validated by the detection of RT-PCR. The expression levels of Pdx-1and Ngn3are even higher in the polycistronic group.(p<0.01),MafA also revealed high level expression in polycistronic group, in the meantime, slightly expressed in the bicistronic group(no MafA introduced). Ins2is also detected positive expression and the expression in polycistronic group is higher (p<0.01).3. Indirect Immunofluorescence test was held3days after transfection. encoded protein of Pdx-1,Ngn3,MafA were all detected positive in each transfected group. And InsulinA was also validated5days after transfection.[Conclusion]1. We successfully constructed the eukaryotic overexpressed plasmids of pcDNA3.1(+)-mafa ORF+ngn33’UTR+pdx13’UTR and pcDNA3.1(+)-ngn3ORF+pdx13’UTR which can express the genes of Pdx-1,Ngn3and MafA in the cells of mice fetal liver.2. With the eukaryotic overexpressed plasmids of pcDNA3.1(+)-mafa ORF+ngn33’UTR+pdx13’UTR and pcDNA3.1(+)-ngn3ORF+pdx13’UTR, we could observe the expression of Pdx-1, Ngn3and MafA both on the transcriptional and translational level, also the islet-related gene Ins2and the protein of InsulinA can also be detected...