Transplantation Of Autologous Induced Multipotent Stem Cells For Treatment Of Chronic Renal Failure

Objective:1. Culturing peripheral blood mononuclear cells in vitro. Using natural active compound (inducing agent) which made in our laboratory to induce the peripheral blood mononuclear cell to the induced multiple stem cells (iMSc),and identify the characteristics of it; 2. To Establish the rabbit model of chronic renal failure by unilateral ureteral obstruction (UUO); 3. Transplantation of autologous iMSc which labeled with BrdU for chronic renal failure. And give the comprehensive evaluation to the therapeutic effect of iMSc.Methods:1.General-level Japanese white rabbits(n=30), the animals randomly divided into two groups according to body mass number:control group (n=10):healthy rabbits without any treatment as control; unilateral ureteral ligation (unilateral ureteral obstruction, UUO) group (n= 20):left ureteral ligation. Left ureteral recanalization after 8 weeks of intervention in the UUO group, then UUO group was divided into two groups:stem cell therapy group (n=10):the model group of animals treated with autologous stem cell transplantation; model control group (n=10):no treatment.2. Culturing peripheral blood mononuclear cells in vitro. Using natural active substances (inducing agent) which made in our laboratory to induce the peripheral blood mononuclear cells into the iMSc and identify the characteristics of stem cell marker genes Oct3/4, Nanog expression by semi-quantitative PCR.3.Labeling the iMSc with BrdU.4. Transplantation of autologous iMSc for chronic renal failure. Detecting blood biochemical indicators of renal function at each time point, and observing the structural changes of renal cell by transmission electron microscop, monitoring of glomerular filtration rate by SPECT. Analysis of renal interstitial fibrosis associated with transforming growth factor (TGF-β1) expression by immunohistochemistry and semi-quantitative PCR. Results:1. the peripheral blood mononuclear cells after induced for 72 hours,can express the Oct3/4, Nanog genes except the peripheral blood mononuclear cells.2. UUO after the first 8 weeks, Scr and BUN increased significantly compare to normal control group (P< 0.01),and Reduced after the iMSc therapy compare to UUO group (P<0.05).3. The GFR in UUO group decreased significantly and glomerular filtration function was lost, the GFR increased after the iMSc therapy,it is higher than the UUO group.4. Immunohisto-chemistry and semi-quantitative PCR showed that TGF-β1 expression had significantly increased in UUO group compare to normal control group (P< 0.05).and reduced after the iMSc therapy compare to UUO group (P< 0.05).5.We can see the significant increase of renal interstitial fibrosis in UUO group and reduce after the iMSc therapy compare to UUO group by transmission electron microscop.6. The best concentration of BrdU for labeling iMSc is 30μmol/L for 72 hours,the amount of labeled positive cells is 85%-90%.Conclusion:1. Natural active compound induced lymphocytes into iMSc.which expressed stem cell specific marker genes Nanog,Oct3/4.2. Stem cell transplantation treatment after 4 weeks, serum creatinine, blood urea nitrogen levels are lower than the model control group; glomerular filtration rate and renal blood flow to a certain degree of recovery; the level of renal fibrosis reduced.3. iMSc with BrdU marking transplanted 4 weeks later,are distributed in the kidney medulla.