Expression Of HBMP7 Gene Carried By Bone Marrow Derived Mesenchymal Stem Cells In Vitro Protect Renal Tubule Epithelial Cells Against Injury Caused By Hypoxia-reoxygenation

BackgroundIschemic acute renal failure caused by ischemical reperfusion injury affects the fuction of transplanted kidney. On account of ischemia-reperfusion associated with renal transplantation and other kidney diseases causing apoptosis and necrosis of cells in renal tubule and glomerulus,and weaking renal function,the basic and clinic researches about stem cells transplantation and gene therapy have been applied in treatment and prevention of ischemical reperfusion injury in kidney. The stem cells transplantation represents technological innovation of tissue regeneration and reparation. With steadily genetic background, lower immunogenicity and regulating immune function,bone marrow derived mesenchymal stem cells(BMD-MSCs) that are suitable to transplant among different individual of same genera,have brought new hopes for the prevention and cure of various kidney diseases,which depends on the following aspects:the first,because of plasticity of MSCs that is characterized by self-renewal and multi-directional differentiation potentiality,MSCs become the seed cells of tissue-engineering; the second, because MSCs that perceive the changes of local microenvironment in vivo, remove and settle down pathological location in tumor or damaged tissue,MSCs become ideal carrier cells;the third,because MSCs,which are isolated from bone marrow,can be expanded in vitro,are easily obtainable and differentiated into several kinds of cells through induction or stimulus of optimal condition in vitro or in vivo. Both acute and chronic renal injury are associated with substantial decrease in the expression of bone morphogenetic protein7(BMP7) that is essential for the conversion of epithelia from condensing mesenchyme during kidney development, enhances the repair of tubular structures in the kidney. In this setting, BMP7 inhibits epithelial-mesenchymal transition involving adult renal epithelial tubular cells. Several reports unequivocally demonstrated that administration of exogenous recombinant human BMP7 enhances recovery of the kidney in animal models of acute and chronic renal injury. Thus,we pay close attention that the transplantation of MSCs after BMP7 gene transfection in vitro protects against ischemical-reperfusion injury in kidney.ObjectiveIn order to simulate ischemical reperfusion injury in kidney in vitro,to establish the model of hypoxia-reoxygenation that deals with human kidney proximal convoluted tubule epithelial cells HK-2, survey and approach the action and mechanism that administration of MSCs culture medium protects HK-2 cultured in vitro against injury caused by hypoxia-reoxygenation after human bone morphogenetic protein7(hBMP7) gene transfection mediated by recombinant adenovirus vector to rabbit MSCs.Methods1. To isolate, purify and amplify MSCs from rabbit bone marrow by density gradient centrifugation and adherent culture in vitro, observe the characteristics of MSCs morphology by inverted phase contrast microscope and transmission electron microscope; survey and map MSCs growth curve; detect cell cycle by flow cytometer; MSCs were induced into osteogenetic cells in the osteogenesis supplement medium or adipose cells in adipogenesis supplement medium, and then they were verified.2. To cut the plasmid of pBluescript sk(+)-hBMP7 including hBMP7 gene by adequate restriction enzyme and construct the shuttle plasmid of pDC316- hBMP7. pDC316- hBMP7 and the skeleton plasmid of pBHGloxΔE1, 3Cre were used to cotransfect HEK293 cells,which are mediated by lipofectamine. The recombinant adenovirus Ad-hBMP7 was obtained through homologous recombination in HEK293 cells. To determine virus titer,optimal multiplicity of infection(MOI) and transfection efficiency. To detecte the expression of hBMP7 in MSCs and culture medium by the methods of RT-PCR,Western blotting,ELISA and Immunocytochemical stain.3. To divide HK-2 into 4 groups,Group A:HK-2 are cultivated under the common culture condition;Group B:HK-2 are cultivated through hypoxia-reoxygenation;Group C and D:after hypoxia,reoxygenation begins with administration of MSCs culture medium, MSCs are infected by Ad-BMP7 in Group C, MSCs are not infected by Ad-BMP7 in Group D. To survey the sum of survival cells and the rate of cell death by MTT colorimetric method, analyze the percentage of cell apoptosis or necrosis by flow cytometry,survey SOD total vigor and MDA content, detect the expression of Bcl-2 by Immunocytochemical stain.Results1. The primary cells and passage cells are mostly fusiform in shape, to be similar to fibroblast cells. The growth curve showed the growth regularity of the passage cells is similar; the cell cycle analysis showed that more than 85% cells were in G0/G1 phase; after MSCs were induced, cell alkaline phosphatase stain showed that alkaline phosphatase is positive in osteogenetic cells, and they formed mineralization nodus; oil red O stain showed that red lipid droplet existed in adipose cells.2. Ad-hBMP7 was obtained through homologous recombination in HEK293 cells and was proved to be correct through PCR,Immunocytochemical stain and ELISA. The virus titer of the prepared Ad-hBMP7 is 7.94×1010IU/ml, which are surveyed by the method of TCID50 and could be used to transfect in vitro or in vivo. When optimal MOI is 100,transfection efficiency is above 90%. hBMP7 measured with ELISA exist in the MSCs culture medium after Ad-hBMP7 transfection. The expression of hBMP7 protein and mRNA was measured through Western blotting,Immunocytochemical stain and RT-PCR.3. After hypoxia-reoxygenation, compared Group A, the sum of survival cells decreased, the rate of cell death and percentage of cell apoptosis or necrosis ascended in Group B,C and D; SOD total vigor decreased, and MDA content increased in Group B,C and D; the expression of Bcl-2 is positive in Group B,C and D. In the same time,the sum of survival cells in Group C or D is higher than that in Group B, the sum of survival cells in Group C is higher than that in Group D;the rate of cell death and percentage of cell apoptosis or necrosis in Group C or D is lower than that in Group B,the rate of cell death and percentage of cell apoptosis or necrosis in Group C is lower than that in Group D; SOD total vigor in Group C or D is higher than that in Group B, SOD total vigor in Group C is higher than that in Group D; MDA content in Group C or D is lower than that in Group B, MDA content in Group C is lower than that in Group D;the expression of Bcl-2 in Group C or D is higher than that in Group B, the expression of Bcl-2 in Group C is higher than that in Group D. Conclusion1. MSCs can be isolated and cultured by the method of density gradient centrifugation and adherent culture in vitro,the better potentiality of proliferation and multi-directional differentiation can meet the requirements of the further experiments.2. The recombinant Ad- hBMP7 was successfully constructed, which could transfect MSCs in vitro. The effective expression of hBMP7 was obtained in MSCs and culture medium.3. Administration of MSCs culture medium after hBMP7 gene transfection mediated by recombinant adenovirus vector to rabbit MSCs and the common MSCs culture medium protect HK-2 against injury caused by hypoxia-reoxygenation,the protective action is associated with MSCs secretion and existence of hBMP7 that is specially obvious.