Establishment Of Rabbit Models Of Renal Interstitial Fibrosis And Treatment Effects Of Induced Autologous Stem Cells

Objective1. To establish rabbit models of renal interstitial fibrosis, and study their kidney's function and histopathological change.2. Using induction agents created in our laboratory induce rabbit skin fibroblast cells into pluripotent stem cells.3. Observation of induced autologous stem cell transplantation in the rabbit of renal interstitial fibrosis distribution, and through the change of serum renal function, SPECT glomerular filtration rate and renal structural analysis , evaluation of induced autologous stem cell transplantation for treatment of renal interstitial fibrosis.Methods1. establish rabbit models of renal interstitial fibrosis: from 25 general-class Japanese , randomly divided into normal group (5): no treatment; model group (20): left ureteral ligation,on the first 8 weeks analyzing plasma creatinine and blood urea nitrogen level and then randomly selected two rabbits were sacrificed for pathological section modeling the situation observed, suggesting that the standard will meet the model were randomly divided into model group: transplant group (10), left ureteral obstruction caused renal interstitial fibrosis transfusion of autologous stem cells after induction; control group (8), left lateral ureteral obstruction caused renal interstitial fibrosis treated by infusion of normal saline treatment.2. The culture, tags and induction of autologous fibroblasts: Rabbit of the transplantation model were remove leg hair, collected skin tissue after disinfection, get skin fibroblast cells adhered to the wall, When the primary cell fusion up to 80% for passage and join carrying GFP retroviral vector (from the lab), GFP labeled medium was changed after 72 h using flow cytometry and transfection efficiency, after 3-4 days the transfer 3rd generation, When the adult cell of the 3rd generation density reached the cell fusion 80% , begun to increase induction medium, after72 h, cells were collected and observed morphological characteristics and cell growth characteristics under inverted microscope.3. Efficacy evaluation of the induced autologous stem cell transplantation for treatment of renal interstitial fibrosis in rabbits: induced autologous stem cell markered by the GFP gene were transplantated into renal artery implantation model animal kidney, after transplantation 1,4,8 weeks, peripheral collection separation of blood serum indicators of renal function related to detection, and after transplantation 8 weeks, renal biopsy, histopathological sections, the tissue sections were observed under fluorescence microscope placed on the distribution of transplanted cells; another small part of the renal tissue were routine pathological sections and HE staining, and observed renal interstitial fibrosis changes in organizational structure after cell transplantation;and changes in glomerular filtration rate in the 16 weeks , using single photon emission computer tomography apparatus (SPECT) scanning.4. Statistical analysis: Experimental data was described as x±s, and SPSS15.0 software used on plasma levels of renal function between the two groups , using independent sample t test, when test value P <0.05 was statistically significant.Result1. . Histopathological changes in rabbit renal model: the model group to take before living kidney biopsy shows severe atrophy of renal tubular reduce the number of tubular basement membrane thickening, structural disorder and the expansion of glomerular cysts, cysts clear the ball inside the capillary occlusion showed ischemic changes, diffuse interstitial inflammatory cell infiltration and severe fibrosis.2. Blood biochemical parameters of the model: before modeling to 8 weeks after modeling, rabbit of the model peripheral blood creatinine levels ranging from (9.30±1.27) mmol /l increased to (14.94±2.06) mmol /l; urea nitrogen levels ranging from ( 99.60±6.73) umol /l increased to (178.00±18.19) umol /l.3. Morphology observation of cell: Primary rabbit skin fibroblasts were adherented after 2-4 hours, 1-2 days the cells became spindle, forming colonies of various sizes, cultured for 3 - 4 days to reach 80% of cell fusion, after passage, evenly distributed, GFP labeled cells were green fluorescence, after 72 hours , morphology of induction cell from the spindle to irregular shape, larger.4. Induced cells in transplantation and implantated cells were observed: the induced cells were transplanted from the rabbits' femoral artery to renal artery, after 8 weeks the organization of renal interstitial fibrosis can be observed green fluorescent cells,that were more gathered in the tubular, glomerular capillary loops, and small arteries. No green fluorescent cells distribution in the control group.5. Histopathology observed after cell transplantation: the transplantation model can be saw moderate renal biopsy shows tubular atrophy, basement membrane thick side, a small tube multifocal expansion, renal cyst expansion, renal interstitial infiltration of inflammatory cells scattered, light fiber of change; Control group with severe renal atrophy, tubular atrophy wide, reduce the number of diffuse interstitial infiltration of inflammatory cells and severe fibrosis.6. Renal function after cell transplantation: after induced autologous stem cell transplantation cell transplantation group serum Scr by the (14.75±1.87) mmol /l reduced to (10.06±2.12) mmol /l, Bun by a (185.30±21.66) umol /l down to (113.78±12.78) umol /l, the control group without cell transplantation Scr by the plasma (15.06±2.24) mmol /l increased to (17.66±3.20) mmol /l, Bun by a (178.40±20.35) umol /l increased to (202.20±23.95) umol /l,transplantation group and control group, 12,16 in the first week Scr, Bun were significantly different (P <0.05).7. SPECT results showed that transplanted group and control group, glomerular filtration rate (GFR) were 67.1ml/min and 47.3ml/min, there are significant differences between the two (P <0.05).Conclusion1. After 8 weeks unilateral ureteral obstruction, successfully established a rabbit model of renal interstitial fibrosis: SPECT detection, histological observation and kidney function and related biochemical analysis results are consistent with the standard of renal interstitial fibrosis.2. Successfully the skin fibroblasts of rabbit renal interstitial fibrosis were markered GFP gene, marker-positive rate was 92%.3. Using inducer created by the laboratory the successful induction of rabbit skin fibroblast cells into pluripotent stem cells. 4. Induced autologous stem cell transplantation can repair damaged parts of the kidney, inhibit the development of renal interstitial fibrosis, partly reverse the process of renal interstitial fibrosis, to the development of the normal structure and improve renal function.