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Background: Diabetes has become second only to cardiovascular disease and cancer death disease, it is a condition that affects insulin and sugar content of the disease, a total of two main types, type I and type Ⅱ The common feature of islet beta cell damage or can not produce enough insulin or the body can not effectively use insulin, so insulin is used clinically as medicines for diabetes. At any time require insulin to patients brought great inconvenience, and international patients with diabetes, islet transplantation and gene therapy research has become a hot spot. But the main problem is the lack of donor immune rejection, the insulin gene transfection efficiency and expression regulation. Human umbilical cord sources of mesenchymal stem cells may be easier to obtain and purified, and easy-to-gene modified, the exogenous gene can be transfected mesenchymal stem cells to create a safe gene therapy of the cell carrier. Studies have shown that mesenchymal stem cells as well as immune suppression, it not only can inhibit the in vitro proliferative response of T lymphocytes in vivo experiments showed similar immunosuppressant, so use it as a cell carrier can avoid immune rejection problems . And the method we use recombinant proinsulin gene transformation so that it can be highly expressed in non-β-cells (mesenchymal stem cells). I believe that our research can provide strong experimental basis for the treatment of type I diabetes. Objective: The human proinsulin gene a mutation in two parts, by adenovirus-mediated transfection into human umbilical cord derived mesenchymal stem cells, study of their ability to express human insulin secretion. Method: The method by RT-PCR amplified from health aborted fetuses pancreatic tissue the pINS cDNA sequence overlap extension PCR method in the two parts of the junction of the A-chain-C peptide and C-peptide-B chain pINS gene. mutation, resulting Furin protease cleavage site. AdEasy TM system the recombinant adenovirus pAdINS, pAdINS-M2 and pAdGFP. Isolation and culture of human umbilical cord mesenchymal stem cells, and its growth characteristics, immune phenotype, differentiation capacity and expression of Furin protease identified. the pAdINS, pAdINS-M2, and pAdGFP infected human umbilical cord mesenchymal stem cells using fluorescence microscopy and flow cytometry detection efficiency of their infection, and to determine the optimal multiplicity of infection (multiplicities of infection, MOI). The multiplicity of infection (MOI) 100 pAdINS, PBH days the pAdINS-M2 or pAdGFP infection of human umbilical cord mesenchymal stem cells, RT-PCR detection of human proinsulin gene expression in human umbilical cord mesenchymal stem cells; Western blot to detect expression of mature human insulin and human C-peptide expression in human umbilical cord mesenchymal stem cells; immunofluorescence assay infection 48 hours after human insulin in human umbilical cord mesenchymal stem cells: ELISA assay after infection 1,3 5,7,10 Heaven insulin and human C-peptide secretion. Results: amplified since health aborted fetuses pancreatic tissue by the pINS 364 bp cDNA ligated into the shuttle plasmid, recombinant adenovirus. titer of purified amplified pAdINS-M2 the 2.57 × 10 PFU / mL, titer of pAdINS is 1.25 × 10 10 PFU / mL, control virus AdGFP as 4.85 × 10 9 PFU / mL. Cultured mesenchymal stem cells isolated from umbilical cord tissue, growth curve, amplified generation in about three days. Cell cycle, most of the cells in the G0-G1 phase (94.39%), and a small part in the G2-M phase (4.15%) and S phase (1.45%). Induced culture conditions, the isolation and culture of human umbilical cord mesenchymal stem cells are able to differentiate into fat cells and osteoblasts. Detected by RT-PCR in human umbilical cord mesenchymal stem cells the Furin protease existence, shows that we construct containing the Furin protease cleavage sites proinsulin gene in the cells can be cut to produce the mature human insulin. By the recombinant adenovirus pAdINS, the pAdINS-M2 and pAdGFP different multiplicity of infection, infection of human umbilical cord mesenchymal stem cells using fluorescence microscopy and flow cytometry infection efficiency, to determine MOI 100 multiplicity of infection. MOI = 100, pAdINS, pAdINS-M2, or pAdGFP infection of human umbilical cord mesenchymal stem cells PBH days later, RT-PCR was used to detect infections in pAdINS pAdINS-M2 human UC-MSCs insulin Gene Expression; Western blot results of UC-MSCs express pAdINS infection infection UC-MSCs in both human proinsulin, UC-MSCs pAdINS-M2 infection detected human insulin and C. peptides, pAdGFP, expression was not detected. The immunofluorescent also detected 48 hours after infection pAdINS-M2 human insulin and C-peptide in human umbilical cord mesenchymal stem cells cytoplasm expression: ELISA detection of infection the PBH-day culture supernatants human insulin and the concentration of C-peptide. The results showed that, since 24 hours after infection of target gene expression, expression reached its peak in the first 3-5 days a week after expressing declined. Consistent with the RT-PCR and Western blot results. Conclusion: We successfully constructed containing the Furin protease cleavage sites of proinsulin gene recombinant adenovirus the body (pAdINS-M2); isolated and cultured human umbilical cord mesenchymal stem cells, and their identification; recombinant adenovirus pAdINS-M2 infection human umbilical cord mesenchymal stem cells, the expression of the secretion of mature human insulin and C-peptide. Can provide strong experimental basis for the treatment of type I diabetes.
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