Microencapsulated Recombinant Hil-10 Gene Cell Lines Constructed

Type I diabetes mellitus is a worldwide populated disease that severely endangered people's healthy nowadays. There are about 2 -5 percent of world population suffering from this kind of disease which is increasing with the time. Most of the patients are children and adult. The cure of type I diabetes mellitus underwent a long course that from drugs to insulin and from pancreatic transplantation to islet transplantation。At present, transplante the microencapsulated islets to treat diabetes is a hot research area. But the inflammatory factors can permeate through the microcapsule and hurt the graft, which result in the die of the transplanted islet.IL-10 is an acidic protein consisted of two homologous subunits. It is called Cytokine Synthesis Inhibiting Factor because of its anti-inflammatory and inhibiting effect to cytokine synthesis. Many experiments have confirmed that IL-10 possessed anti-inflammatory and immunosuppressive activity. Act as down-regulation (repressive) factor, IL-10 can inhibit immunoproliferation and inflammatory reaction.Based on the above reasons, via constructing a genetic engineering cells which express hIL-10 steadily,then microencapsulated it. my research focuses on treating diabetes by islets transplantation through combining hIL-10 and microcapsulation.Methods:(1) Prepare the microcapsules and evaluate the quality of the microcapsules, (2)obtain human IL-10 cDNA fragment by RT-PCR from peripheral blood lymphocyte cell mRNA; add the signal peptide derived from the rattus albumin protein to 5'end of ML-10 cDNA; clone the recombination cDNA fragment into pcDNA3 to construct the eukaryotic expression vector pcDNA3-hIL-10(3)transfect the c2c12 cell with pcDNA3-hIL-10. transgenic clones are screened by G418. (4) microencapsulate the transgenic cells and detect the expression of hIL-10 through western-blot; detect the growth of the cell by MTT.Results: (1) The constructed APA microcapsules possessed excellent mechanical intensity, biocompatibility and permeability. (2)The sequence of hIL-10 was identical with published sequence (NCBI gi:24430216 ). the vector pcDNA3/rAlbs-hIL-10 was performed. The sequence analysis results show that there is 96% homology between the rat albumin signal peptide cDNA and the reported rat albumin signal peptide cDNA in Genebank, while the hIL-10 cDNA was consistent with the reported cDNA in Genebank, but Amino acids are identical. (3) ML-10 was successfully expressed in cell conformed by RT-PCR, western-blot and immunohistochemistry. (4) after three days, ML-10 could be detected in culture supernatant, this results suggested that ML-10 could permeate through microcapsule.conclusion: The eukaryotic expression vector of hIL-10 was successfully constructed, and obtained the microencapsulated genetic engineering c2c12/hIL-10 cell strain ,which can express ML-10 steadily. After being microencapsulated, the cell can continue to proliferate.