Construction Of Microencapsulated Cell Expressing Human Vascular Endothelial Growth Factor

Context:The problem of vascularization in tissue engineering is thelimiting-speed-step in tissue engineering.Revascularization is so important forthe regenerated tissue and organ that they can get adequate oxygen andnutriments through the metabolic pathway and then produce a markedbiological effect into the bodies of host. Otherwise the biological effect wouldnot be produced and the state of an illness would be worse because of thesiltation of metabolite and the necrosis of tissue and organ.For this reason , itis the most important that how to solve the problem of vascularization intissue engineering.In 1989, Ferrara et al.isolated the VEGF from the culture medium ofpituitary follicular cells first.It is a mitogen exclusively for endothelial celland have the effect of inducing vasculature intensively and increasing thepermeability of veinule and small vein.And now, it is used in three formsbelow primarily: as the form of proein;transfect the subject cell by liposomeor viral vector;as the form of genetically engineered cell. The geneticallyengineered cell is transfer the exogenous gene into the subject cell utilizing allkinds of methods of gene transfer to make the cell expressing and secretingcorresponding protein.We hope to solve the problem of vascularization intissue engineering through constructing the genetically enginerred cell ofhVEGF165.To solve the problem of immunological rejection we combinedmicroencapsulation with genetically enginerred cell to construct themicroencapsulated genetically enginerred cell expressing hVEGF165steadily.The definition of microcapsule is the global particle made of highmolecular membrane structure encapsulating liquid or gas , diameter range1 ～ 5000μm.The process of producing microcapsule is calledmicroencapsulation. T.M.Chang rase the idea of constructing biotic artificialorgans by microencasulation first.Allogeneil graft and xenotranslantation canbe performed with tissues or cells possessing specified effect under theimmunoprotection of the membrane facultative permeability of themicrocapsule without immunosuppressive agent.The bioactive substance wassupplemented, and then we answered our purpose of curing disease.We hopeto solve the problems of immunological rejection and vascularization withoutimmunological rejection or slightly in tissue engineering through constructingmicroencapsulated genetically engineered cell expressing hVEGF165 steadily.Objective:This study was planed to construct a microencapsulatedgenetically engineered cell expressing hVEGF165 steadily.We want to use itin organs reconstruction or transplanting into ischemic tissues or organsdirectly in the field of tissue engineering.Methods:First, we designed a pair of specific primers according thehVEGF165 sequence published in the GenBank that we have knew,namedP1,P2, and the clone sites of BamHⅠ,XhoⅠ were introduced respectively intothe upstream and downstream primers.We made 0.5g human embryo livergrinded to powder in liquid nitrogen and got out the total RNA and then thehVEGF165 cDNA was cloned with RT-PCR.We cloned the cDNA intopGEM-T vector, named pGEM-T-hVEGF165.PCR was performed to makesure the direction that the cDNA was cloned with the primers T7 the vector'sautospecific primer and P2.we designed before.The positive clone was sent toDNA sequence after a double enzymes digested assay.To construct theeukaryotic expression vector of hVEGF165,we subcloned the cDNA into theeukaryotic expression vector pcDNA3.The PCR and double enzymes digestedassay were performed and the positive clone was sequenced as before.Underthe effect of Lipofectamine 2000, the pcDNA3-hVEGF165 was introductedinto the cells c2c12.There three groups in this experiment: in the experimentalgroup, pcDNA3-hVEGF165 was introducted into c2c12 cells;in the controlgroup1 pcDNA3 was introducted into c2c12 cells;in the control group2nothing was introducted into c2c12 cells.The cells were moved into T25 cultreflask and G418 was added at the concentration of 200μg·ml-1,400μg·ml-1,600μg·ml-1,800μg·ml-1,1000μg·ml-148 hours. post transfection cultured 7to 10 days.We picked single clone cell from the survival cells with limitingdilution assay.Then RT-PCR and Western blot were prerformed to exame theexpression of hVEGF165. With the technique of microencapsulation weencapsulated the cell steadily expressing hVEGF165 with alginate to protectthe cells from the attack of immune systems. Also we examined theexpression and release of the hVEGF165 from the encapsulated cell withWestern blot and hVEGF165 ELISA kit.Results:The DNA sequencing of pGEM-T- hVEGF165 and sequenceblast shows we cloned the hVEGF165 cDNA from the human embryo liver,coincidence with the sequence AB021221 published in GenBank, total length576bp including signal peptide and mature peptide.The cDNA of hVEGF165 was directed cloned into pcDNA3.The positiveclone was sent to sequence after PCR and double enzymes digestedidentification.The results of DNA sequencing and sequence blast shows thecDNA was cloned into pcDNA3 in the right direction and the sequence isright. Under the effect of Lipofectamine 2000, the pcDNA3-hVEGF165 wasintroducted into the cells c2c12 and the G418 was added into the culturemedium 48 hours post transfection.The cells in control group2 appeared death48 hours after adding G418.The cell number of death increased as theconcentration of G418 had increased.9 days after adding G418, the cells incontrol group2 were dead completely at the concentration of 800μg·ml-1 and1000μg·ml-1.So 800μg·ml-1 was taken as the concentration of the experimentgroup and control group1 and then the concentration of G418 was cut down to200μg·ml-1 and kept . After picking single clone cell, RT-PCR and Westernblot were prerformed to exame the expression of hVEGF165.The resultsshows that the genetically cell we produced can express hVEGF165 on bothRNA level and protein level.At last the cell was encapsulated with alginate, the diameter of themicrocapsules was about 500~550μm.We also detected the expression ofhVEGF165 in the culture medium.The quantity of hVEGF165 in the culture medium was measured withhVEGF165 ELISA kit.The hVEGF quantity of microencapsulated cells inculture medium was 300.87±32.62pg per 1.5×106cells,;the quantity ofunencapsulated cells in cuture medium was 1199.65±102.33 pg per 1.5×106cells.The results of in vitro test of cell proliferation shows thatmicroencapsulation would not inhibite cell proliferation, but a hysteresisobserved in the beginning of cell proliferation.Conclusion: We constrcted a microencapsulated cell line that couldexpress hVEGF165 steadily and defend themselves from the attack of largeimmune molecule for the treatment of ischemic illness and vascularature inthe field of tissue engineering as a tool.