Pancreas Specific Expression Vector And The Expression Of PDX1-Hes1 Validation Study

Organ transplantation is regarded as the effective way to supply human organ and to save human lives.But various patients face the serious shortage of organ donors.With the depth of the scientific research and the improvement of science and technology,xenotransplantation has gradually become a hot topic in the field of medical research.Because of the size and the blood physiological and biochemical indexes are more close to human,as well as the growth cycle is short,so pigs can be used as the preparation of special organs,which are becoming the best provider of xenotransplantation donor.Along with the development of transgenic technology and induced pluripotent stem cells(iPS),pigs were believed to be a reliable source of donor organ transplantation.Diabetes and pancreatic cancer are caused by pancreatic organ failure.On the demand of pancreas transplantation in diabetes and pancreatic cancer grow,the pancreas transplantation is also facing a serious shortage of donor and matching difficulty.Interesting that by using iPS and human-pig chimeric technology,it can achieve a personalized organ transplantation with the reduced immune rejection.It is important firstly to prepare the pig embryo defective pancreas.PDX1 plays an important role in the growth and development of pancreas.Notch signaling pathway plays an important regulatory role for early pancreas development and pancreatic cell fate decisions.In addition,through transcriptional inhibition of cell proliferation and differentiation during embryonic development period,Hes1 Notch signaling pathway regulate the downstream of transcription,In this study,the pancreas-specific PDXl-eGFP and PDX1-Hes1 vectors were constructed.These two vectors were transfected on ? cells in mice to validate the carrier-specific expression in vitro.Transgenic mice were produced with PDX1-eGFP and PDX1-Hes1 two vectors to verify the specificity of the expression in vivo,and further to establish the animal models of pancreatic defect.The main results are as follows:Experiment 1:Construction and verification of PDX1-eGFP and PDX1-Hes1 vectors.Fibroblast cells and p cells were transfected with PDX1-eGFP vector for 48 h.Subsequently,fluorescence can be observed on ? cell but not on fibroblast cells.In addition,Western Blot analysis was performed both on these cells and the results show that eGFP protine can be observed in ? cells but not in fibroblast cells;PDX1-Hes1 and empty vector were transfected into mouse pancreatic ? cells,after 48 h,real-time PCR and Western Blot analysis were performed.Results showed,Hes1 expression levels in PDX1-Hes1 transfected ? cells was significantly higher than the empty vector transfected ? cells.It is confirmed that Hes1 gene specific over expressed in pancreatic cells.Experiment 2:Production of transgenic mice with PDX1-eGFP and PDX1-Hes1.We successfully produced transgenic mice that one with PDX1-eGFP and two with PDX1-Hes1.Subsequently,RT-PCR and Western Blot analysis were performed on these transgenic mice.Results show that,eGFP expressed in the liver of PDX1-eGFP mice and fluorescence were observed in PDX1-eGFP mouse pancreas parts in vivo.In addition,Hes1 over expressing in mice with PDX1-Hes1.Although no defective pancreas was not observed in PDX1-Hes1 mice,but a certain degree of pancreatic abnormalities were observed.