| BACKGROUND Chronic kidney disease(CKD)increasingly become international public health problem.Although renal replacement therapy(RRT)achieved great progress in treatment of CKD,RRT cannot be used to clinical because the mortality of patients who had received RRT is still up to 21%～23%and source of donor kidneys were deficiency,tissue match was difficult,a high charges,rejection of renal transplantation failed to thoroughly control.The pathogenesis of CKD was progressive interstitial fibrosis and the main morphological change of fibrosis was excessive accumulation of extracellular matrix.Reversing interstitial fibrosis was common pathway of treatment of CKD.Gene transfer for treatment of organs or tissues fibrosis was research focus in chronic disease.There were many studies on curative effects of uPA on liver fibrosis or cirrhosis and pulmonary fibrosis in china and abroad.But no significant effect of gene theraputic on fibrosis was observed.Renal fibrosis was not merely excessive accumulation of extracellular matrix but important ECM could damage near cytoarchitecture leading to nephrons reducing.Even ECM were degradated by uPA,renal function was uncertainly improved without enough functional nephrons.Clinical effects would be obvious when fibrosis were reversed before cell death or reconstructed complete and functional nephrons at the same time. Transplantation of stem cells for the treatment of kidney disease has become focus in chronic disease field because stem cells had self-renewal potential and differentiation capacity.Metanephric mesenchymal contained stem cells of embryonic kidney.Metanephric mesenchymal had potential of oriented development and could differentiate into nephrons under proper induction.Metanephric mesenchymal could develop renal-like organs which had nature renal's structure and ultramicro-structure.The mature nephron and collecting duct from metanephric mesenchymal had no difference with nature renal under light microscopy and electron microscopy.Up to now the study was not reported whether metanephric mesenchymal could repair chronic kidney disease.Even stem cells could home to lesion location,its ability of differentiation and rageneration could be limited because ECM occupied spaces of cell regeneration.So got an idea that metanephric mesenchymal were used as gene carrier to improve expression of uPA which degradated ECM,then more metanephric mesenchymal could home to lesion location in order to promote recovery of renal function.â‘ Whether metanephric mesenchymal transplantation in rats with UUO have renal protective effect.â‘¡That transfer of uPA gene into metanephric mesenchymal targeting gene to lesion location using the homing characteristics of stem cells will improve the expression of uPA,degradate ECM,repair injury renal cells and regenerate renal cells.â‘¢Obsever the change of renal function and renal fibrosis association protein,trace the homing and differentiation of metanephric mesenchymal and study the protective mechanism.Combination of cell therapy and gene therapy aims to explore and study new ways of preventing the progression of chronic kidney disease.OBJECTIVE Study of regeneration role and possible mechanism of metanephric mesenchymal and metanephric mesenchymal transfected by uPA gene in UUO rats.METHOD1.Construction and package of recombinant plasmid of uPA (urokinase-type plasminogen activator)gene and pAAV-IRES-hrGFP vector.(1)The uPA gene fragments were amplified by reverse transcription and polymerase chain reaction from total RNA of rat kidney. T4 DNA ligase catalysed uPA fragments cloning into BamHâ… and Xhoâ… sites of multiple cloning sites of pAAV-IRES-hrGFP plasmid and got recombinant plasmid of pAAV-hrGFP-uPA.(2)The recombinant plasmids of pAAV-hrGFP-uPA were verified whether they have uPA fragments by digestion of BamHâ… and Xhoâ… and whether pAAV-hrGFP-uPA sequence was correct by DNA sequence analysis. (3)The recombinant plasmids or AAV plasmid were transfected into the AAV-293 cells together with the control plasmids pHelper and pAAV-RC by phosphate-calcium deposit method and got rAAV viral particles and AAV viral particles,respectively.(4)The titer of viral particles was determined by dot hybridization.(5)Viral particles transfected AAV-293 cells again to detect its infectious potential.2.The viral particles of rAAV and AAV were transfected into rat's metanephric mesenchymal(rat-inducible metanephric mesenchymal-18, RIMM-18)in vitro and the positive RIMM-18 cells that expressed GFP were detected using the inverted fluorescence microscope.(1)The viral particles of rAAV and AAV were transfected into RIMM-18 in vitro when MOI=1×105.(2)The positive RIMM-18 cells expressed GFP were detected using the inverted fluorescence microscope after 48h.(3)The transfected RIMM-18 cells were passaged.(4)Identification of transfected RIMM-18 cells were implemented by HE staining,Giemsa staining and immunohistochemical staining of vimentin and cytokeratin. (5)Transfected RIMM-18 cells were detected by RT-PCR and Western blotting techenique.3.Study of regeneration role and possible protective mechanism of metanephric mesenchymal and metanephric mesenchymal transfected by uPA gene in UUO rats that were transplanted via caudal vein. (1)Unilateral ureter of two hundred female Sprague Dawley rats aged three months were ligated to estabilish UUO model and randomly divided into five groups:model group,culture medium group,rAAV group,AAV group and RIMM-18 group.At the same time,rats of culture medium group were injected culture medium via caudal vein,rats of rAAV group were injected RIMM-18 cells transfected by rAAV viral particles,rats of AAV group were injected RIMM-18 cells transfected by AAV viral particles and rats of RIMM-18 group were injected RIMM-18 cells.(2) At 7d,14d,21d,28d,the urine and blood of rats were collected to examine urine protein in 24h,blood urea nitrogen,serum cretinine,serum albumin and endogenous creatinine clearance rate.The kidney of all rats were collected to have pathological analysis and observe the expression of uPA,PAI-1 and TGF-β1 through technique of immunohistochemical staining,real-time PCR and Western blotting.(3)At 1.5d,3d,7d,14d,21d, 28d,the obstructed kidney,contralateral kidney,liver,spleen,heart and perpheral blood of rAAV group and AAV group were collected to observe GFP using the inverted fluorescence microscope.RESULT1.(1)uPA cDNA obtained from immature rat kidney by reverse transcription polymerase chain reaction method were correct.(2)The results suggested the recombinant AAV vectors carrying uPA gene were constructed successfully.The sequence of the recombinant plasmid of pAAV-hrGFP-uPA were proved identical to the reported cDNA sequence by DNA sequence analysis.(3)The viral particles titer of recombinant AAV and uPA gene were 1.12×1012vg/mL by dot hybridization and AAV were 1.0×1012vg/mL.(4)AAV-293 cells were transfected by rAAV viral particles and AAV viral particles and the ratio of positive cells after 48h were 80%and 95%respectively when the MOI=1×105.2.(1)The ratio of RIMM-18 cells that were transfected by viral particles of rAAV and AAV were 60%.(2)The expression of GFP were decreased with the passage of transfected RIMM cells.(3)The transfected RIMM-18 cells still have the original characteristic and were vimentin positive and cytokeratin negative.(4)There were uPA gene in RIMM-18 cells transfected by rAAV and no uPA gene in RIMM-18 cells transfected by AAV by RT-PCR.(5)There were rio uPA expression in RIMM-18 cells transfected by rAAV or AAV by Western blotting.3.(1)At 14d,21d,28d,renal interstitial injury of rAAV group,AAV group and RIMM-18 group's rats were more relieved than those of model group and culture medium group.(2)Urine protein in 24h,blood urea nitrogen,serum albumin and endogenous creatinine clearance rate were not different between five groups.At 14d,28d,serum cretinine of rAAV group,AAV group and RIMM-18 group's rats were less than those of model group and culture medium group.(3)The expression of uPA by semiquantitive analysis of immunohistochemical staining of rAAV group, AAV group and RIMM-18 group's rats were stronger than those of model group and culture medium group.The expression of PAI-1 and TGF-β1 of rAAV group,AAV group and RIMM-18 group's rats were less than those of model group and culture medium group.(4)The uPA gene by RT-PCR analysis of rAAV group's rats were stronger than those of AAV group,RIMM-18 group,model group and culture medium group.There were no different between rAAV group's rats at 7d,14d,21d, 28d.The PAI-1 and TGF-β1 gene of rAAV group,AAV group, RIMM-18 group's rats were less than those of model group and culture medium group.(5)The expression of uPA by Western blotting of rAAV group,AAV group and RIMM-18 group,s rats were stronger than those of model group and culture medium group.The expression of PAI-1 and TGF-β1 of rAAV group,AAV group and RIMM-18 group's rats were less than those of model group and culture medium group.(6)There were GFP in the renal tissue of rAAV group and AAV group's rats at 1.5d,3d,7d,14d,21d,28d and at the same time there were GFP in the contralateral renal tissue.There were GFP in the liver and spleen tissue at 1.5d,3d and GFP became less at 7d,14d,21d,28d.There were no GFP in the heart tissue and blood smears at every time.CONCLUTION1.The recombinant plasmid of AAV and uPA gene were successfully constructed and packaged.2.The transfection rate of rAAV and AAV viral particles to RIMM-18 was as high as 60%.AAV vector can deliver uPA gene effectively.3.(1)RIMM-18 cells could improve renal function and reduce fibrosis of UUO rats by transplantation via caudal vein.(2)The transferred uPA gene could not express.AAV vector had no effect on RIMM-18 cells.(3)RIMM-18 cells transfected by rAAV and AAV viral particles could home to injured renal by obversing the expression of GFP under the fluorescence microscope.GFP positive cells settled in tubular and RIMM-18 cells could repair injured renal.(4)There were GFP positive cells in contralateral renal,liver and spleen tissue and no GFP positive cells in heart tissue and blood smears.(5)There were no immunological rejection and tumor in cell transplantation groups until 4 w.
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