| Backgroud and objectiveAcute renal failure(ARF) caused by renal ischemia-reperfusion injury(IRI) remains a frequent clinical complication, associated with an unacceptably high mortality rate, in large part due to the ineffectiveness of currently available therapies. Ischemia-reperfusion causes changes in tubular cell polarity and necrotic and apoptotic cell death of tubular cell. The recovery of renal function requires the repair of the damaged tubular cells. Bone marrow mesenchymal stem cells(BM-MSCs), which are isolated from bone marrow mononuclear cells, can be expanded ex vivo and ,not only have the ability to differentiate into many cell types including renal tubular cells, but also contribute to renal parenchymal turnover and regeneration. BM-MSCs may home to the injured kidney where they participate the repopulation and repair of tubules of the injured kidney to promote the structural and functional repair of kidney. Though the BM-MSCs have the effective therapeutic action on renal IRI, the amount of transplanted BM-MSCs is limited because redundant BM-MSCs might increase the risk of embolism in different organs. And the amount of BM-MSCs migrated to injured kidney is less in the bad environment after IRI. So many scientists adopted the combined strategy of cell transplantation and gene therapy to promote the therapeutic action of BM-MSCs and have observed the significantly promoted therapeutic action. The bone morphogenetic protein-7 (BMP-7),which possesses regulating effect on cell proliferation, has important role for kidney development and maintaining renal function. BMP-7 in the kidney reduces obviously after renal ischemia-reperfusion. The application of exogenous BMP-7 can promote renal regeneration and recovery of renal function. So the present study was aimed at evaluate the therapeutic action of the human BMP-7(hBMP-7)-modified BM-MSCs transplantation in a model of renal IRI. Methods:1.The construction of the recombinant adenovirus Ad-hBMP-7To cut the plasmid of pBluescript sk(+)-hBMP-7 including hBMP-7 gene by adequate restriction enzyme and construct the shuttle plasmid of pDC316- hBMP-7. Amplified and purified pDC316- hBMP-7 was used to cotransfect HEK-293 cells with the skeleton plasmid of pBHGloxΔE1,3Cre and lipofectamine and the recombinant adenovirus Ad-hBMP-7 was obtained through homologous recombination in HEK-293 cells. The restriction enzyme cutting, sequencing, ELISA and immunohistochemical staining were applied to identify the constructed plasmid and recombinant adenovirus.2. Ad-hBMP-7 transfecting BM-MSCs in vitroThe BM-MSCs were obtained by the combined strategy of density gradient centrifugation and adherence. Ad-hBMP-7 was used to transfect the cultured BM-MSCs with different MOI, and the morphology and proliferation of the transfected BM-MSCs was observed. The expression of hBMP-7 in BM-MSCs was detected with the methods of RT-PCR, Western Blot, ELISA and immunohistochemical staining.3.The observation of hBMP-7-modified BM-MSCs transplantation in the rabbit with renal IRI①The cultured BM-MSCs was transfected with Ad-hBMP-7 (MOI=100) in vitro ,and then was labeled with Hoechst33342 before transplantation.②To establish the model rabbit of renal cold IRI;③Ninety rabbits were divided into 6 groups. Group I:Sham operation group; Group II:control group of IRI; Group III: Ad-EGFP transfected BM-MSCs transplantation after renal ischemia-reperfusion; Group IV: Ad-hBMP-7 therapy in vivo after renal ischemia-reperfusion; Group V: the combined strategy of Ad-EGFP-transfected BM-MSCs transplantation and Ad-hBMP-7 therapy in vivo after renal ischemia-reperfusion; Group VI:hBMP-7-modified BM-MSCs transplantation after renal ischemia-reperfusion.4.The detection was taken at the timepoints of 3, 7 and 14 days after renal ischemia-reperfusion respectively(n=5). The expression of hBMP-7 gene in kidney was detected with RT-PCR, ELISA and Western Blot. The expression of endogenous BMP-7 and TGF-β1 were detected with RT-PCR and ELISA. The activity of SOD and content of MDA in kidney were detected with colorimetric method. The Hoechst33342 positive cells and double positive cells of Hoechst33342 and CK18 were observed under the fluorescent microscope. To detect Bax, Bcl-2 and PCNA in kidney with immunohistochemical staining. The apoptosis of cells was detected with TUNEL method. The changes of renal histology were observed under the light microscope and transmission electron microscope. The blood Crea and Urea were detected to evaluate the renal function.Results1.The shuttle plasmid of pDC316- hBMP-7 was proved to be correct through restriction enzyme cutting and sequencing. The recombinant Ad-hBMP-7 was obtained through homologous recombination in HEK293 cells and was proved to be correct through ELISA, PCR and immunohistochemistry. The virus titer of prepared Ad-hBMP-7 was 7.94×1010IU/ml by the method of TCID50 and could be used to transfect in vitro or in vivo.2.The optimal MOI was determined to be 100 for Ad-hBMP-7 transfecting BM-MSCs in vitro with the transfecting efficiency of 90.52%. Compared with non-transfected BM-MSCs, no significant change of Ad-hBMP-7-transfected BM-MSCs was observed. And the growth of Ad-hBMP-7-transfected BM-MSCs was not markedly promoted or inhibited. The hBMP-7 protein was measured with ELISA in the culture medium of Ad-hBMP-7-transfected BM-MSCs. The expression of hBMP-7 protein and mRNA was measured through immunohistochemistry, Western Blot and RT-PCR in Ad-hBMP-7-transfected BM-MSCs, but not in non-Ad-hBMP-7-transfected BM-MSCs. The detected results with above-mentioned methods showed that the level of hBMP-7 protein and mRNA reached a peak at day 3 after transfection and remained at this high level until day 7 after transfection, then began to decrease slowly.3.①The method labeling BM-MSCs with Hoechs33342 was reliable. The procedure to establish the model of renal cold IRI was successful and the rabbits after operation lived well.②The expression of hBMP-7 protein and mRNA was measured through ELISA, Western Blot and RT-PCR in kidney of group IV,V and VI. Compared with group IV and V , the level of hBMP-7 protein and mRNA was higher in group VI(P<0.05).③The Hoechst33342 positive cells were not observed in heart, liver, spleen and lung under fluorescent microscope. But the Hoechst33342 positive cells were observed in kidney of group III, V and VI and these cells mainly distributed in the renal tubules of inner zone of cortex and external zone of medulla. The Hoechst33342 positive cells and double positive cells of Hoechst33342 and CK18 in group VI were more than those in group IV and V(P<0.05) at each timepoint.④Compared with group I, the expression of BMP-7 mRNA and protein in group II significantly decreased, the expression of TGF-β1 mRNA and protein significantly increased(P<0.05).Compared with group II～V, the expression of BMP-7 mRNA and protein, the PI of renal tubular epithelial cell, Bcl-2 and ratio of Bcl-2/Bax, the activity of SOD in group VI were maximum(P<0.05); The expression of TGF-β1 mRNA and protein, the AI of renal tubular epithelial cell, Bax, content of MDA, blood Crea and Urea in group VI were minimum(P<0.05). Compared with group II～V, the level of damage in renal histology in group VI was the slightest(P<0.05).Conclusion1.The recombinant Ad- hBMP-7 was successfully constructed and transfected BM-MSCs in vitro. The effective expression of Ad- hBMP-7 protein was obtained by Ad- hBMP-7-transfected BM-MSCs.2.The model of renal cole IRI was successfully established.3.The Ad- hBMP-7-modified BM-MSCs seemed to migrate preferentially to sites of damaged kidney and promoted significantly the expression of hBMP-7 protein in kidney.4.Compared with BM-MSCs transplantation or Ad- hBMP-7 infusion in vivo or the combined strategy of BM-MSCs transplantation and Ad- hBMP-7 infusion in vivo, Ad- hBMP-7-modified BM-MSCs transplantation significantly lessened the level of damaged kidney and promoted the structural and functional repair. The Ad-hBMP-7-modified BM-MSCs transplantation protected significantly against renal ischemia-reperfusion injury through the following mechanisms: To lower the level of lipid peroxidation and enhance the ability to clean up the free radicle; To accelerate the expression of endogenous BMP-7 and cut down the expression of TGF-β1 in kidney; To promote cell proliferation and suppress apoptosis in kidney through up-regulating the expression of Bcl-2 protein down-regulating the expression of Bax protein; To raise the amount of transplanted BM-MSCs in kidney and promote those cells directly participating structural and functional repair.
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