Emperimental Study On Bone Morphogenetic Protein-7 CDNA Engineered Bone Marrow Mesenchymal Stem Cells Differentiation Into Renal Tubule Epithelial-like Cells In Vitro

Objective: To investigate the effect of lentivirus-mediated human Bone Morphogenetic Protein-7 gene cDNA which engineered Rat bone marrow mesenchymal stem cells (BMSCs) on BMSCs differentiate into renal tubular epithelial cells in renal tubular epithelial cells Hypoxia/reoxygenation model in vitro,and investigate the feasibility of gene theraphy combination with stem cells theraphy for the acute kidney injury,which based on BMSCs as gene transfected targed cells.Methods: 1.MSCs were isolated and cultured by the plastic adherence method in vitro and identified by CD29, CD34, CD44, CD45.Growth curve and adhesive rate curve were drawn and biological properties of different passages MSCs were analysed.P3 or P4 populations MSCs which bioactivity was well were choosed as BMP7 gene transfection target cells;2.To construct the GFP-labeled four plasmid system of the lentiviral vector carrying BMP-7 gene (Lv-hBMP-7-GFP),and transfected into MSCs.The changes of cells' proliferation were analyzed by MTT, Brdu incorporation and flow cytometry;3.To construct renal tubular epithelial cells Hypoxia/reoxygenation model in vitro and detect cells apoptosis by flow cytometry.MSCs were co-cultured within transwell system,and collect cells by 3d\5d\7d,detect the expression of protein E-cadherin by RT-PCR,and the expression of Cytokeratin18 were taken through immunocytochemical staining method and flow cytometry.Results:1.Lv-hBMP-7-GFP can be highly transfected into rMSCs,the transfection efficiency were approximately 70%, and there was no significant difference of cell proliferation among the cells transferred or not(P>0.05);2.After BMSCs which were trabsfected and co-culture with renal tubular epithelial cells in Hypoxia/reoxygenation model,the expression of E-cadherin and CK-18 were visibly and enhanced along with co-culture time,and there was significant difference among group single rMSCs,group rMSCs co-culture with nomal renal tubular epithelial cells,group rMSCs which were transfected and co-culture with nomal renal tubular epithelial cells(P<0.01),and there was statistical difference among group rMSCs which were transfected Lv-hBMP-7 and co-culture with H\R renal tubular epithelial cells , compared with group rMSCs which were transfected Lentivirus and co-culture with H\R renal tubular epithelial cells,group rMSCs co-culture with H\R renal tubular epithelial cells,group rMSCs which treatment with BMP7 and co-culture with H\R renal tubular epithelial cells(P<0.05).Conclusions:BMSCs can differentiated into renal tubular epithelial-like cells in renal tubular epithelial cells Hypoxia/ Reoxygenation model in vitro,and recombinant hBMP-7 can promoted the procedure.The consequence maybe provid a new way for gene theraphy combination with stem cells theraphy on the acute kidney repair.