| Objective:To investigate the separation, purification and cell identification methods of bone marrow mesenchymal stem cells (BM-MSC), safety and feasibility of labeling BM-MSC by superparamagnetic iron oxide (SPIO). In order to determine the best infusion way of BM-MSC,we analyze the relationship between the distribution of BM-MSC in vivo and cell infusion way.。Methods:After extraction the bone marrow from Tibetan miniature pig, bone marrow mesenchymal stem cells (BM-MSC) were isolated and purified by the method of density gradient centrifugation, the cell growth characteristics were observed in the light microscope and their growth curves were recorded. Cells obtained were identified by adipogenic, osteogenic induction and surface markers CD45/CD90/CD105, which were detected by flow cytometry. BM-MSC were labeled by SPIO in vitro, the effect of different SPIO concentrations on the viability and proliferation of labeled cells was assayed by MTT method. After Tibetan miniature pig kidney transplantation models were established, SPIO-labeled BM-MSCs were infused separatedly from transplant renal artery and common iliac vein. Organs of the recipient pigs were cut and left for further determination, the distribution of BM-MSC in transplanted kidney, lungs and other organs were observed by biopsies stained by Prussian blue and transmission electron microscopy.Results:High-purity BM-MSCs with good growth vigor and passage capacity can be obtained by application of gradient density centrifugation method; BM-MSC could be induced to differentiate into bone cells and fat cells, calcium nodules and fat particles can be observed in cells by Qian red pigment and oil red O staining. Cell surface markers were detected by flow cytometry, the positive rate of CD90was96.9%,the positive rate of CD105was95.7%, the positive rate of CD45was1.1%(negative). The rate of BM-MSC labeled by SPIO was close to100%.By MTT assay, we found that25ug/ml was the appropriate and effective label concentration, which had less impact on the proliferative capacity of BM-MSC, significant difference could be found in the proliferative capacity of BM-MSC labeled by SPIO with concentration of50ug/ml after2days. Intracellular vesicles containing high-density iron particles could be observed by transmission electron microscopy. Transplanted models of Tibet mini pig kidneys could be successfully established, end to side anastomosis method was applied to renal artery and common iliac artery, renal vein and common iliac vein. After BM-MSC infused separatedly from transplant renal artery and common iliac vein, we had observed BM-MSC distribution in recepient pigs:BM-MSC cells in renal allografts by the infusing way of transplant renal artery were significantly more than by the way of common iliac vein, there was significant difference, P<0.05; the number of cells in lungs increase by the intravenous infusion way, a statistically significant difference could be found. Complications including blood clots, infection and others had not occurred in all recipient pigs after BM-MSC infused by the two pathway.Conclusion:High purity, good growth and vitality BM-MSC can be obtained by density gradient centrifugation; BM-MSC can be successfully identified by cell shape characteristics, induction of differentiation and cell surface markers; The method of labeling BM-MSC by SPIO is simple and feasible,it is easier for trace of cells in vivo; Infusion of BM-MSC from transplant renal artery is a safe and ideal pathway,which is conducive for BM-MSC to plant and differentiate in renal allograft. Objective:To induce immune tolerance by autologous bone marrow mesenchymal stem cells combined with low doses of FK506in living-related donor kidney transplantation; to explore the role and mechanism of induction of transplant immune tolerance by autologous bone marrow mesenchymal stem cells, parameters about immune tolerance will be observed and analyzed such as functions of transplanted kidney, count and rate of lymphocyte subsets, rate of acute rejection and other indicators.Method:The patients with chronic renal failure (uremia) were enrolled in,who would receive living-related donor kidney transplantation at our center within2009.9to2012.9. The patients,enrolled in according to inclusion criterias, were randomly divided into Group control (10cases, without autologous BM-MSC infusion) and Group experiment (10cases, with autologous BM-MSC infusion). Autologous bone marrow mesenchymal stem cells had been prepared1month before transplantation, BM-MSC would be infused three times in total into patients in Group experiment,the first infusion was during transplantationg, with5×106BM-MSC,by the pathway of transplant renal artery; the second infusion (1week post-transplantation) and the third infusion (1month post-transplantation),with1×106/kg/time BM-MSC cells, by the pathway of peripheral intravenous. The initial dose of FK506for Group control was0.08mg.kg-1.d-1, Group experiment0.04-0.05mg. kg-1.d-1, the same dose of Mycophenolate mofetil and prednisone for the two groups. Functions of renal allograft,24-hour urine protein, lymphocyte count and proportion were dynamically observed after renal transplantation, T lymphocyte subsets and proportion, Treg proportion were detected by flow cytometry, procedural biopsy for transplanted kidneys had been carried out to observe the incidence of acute rejection.Results:High-purity BM-MSC had been obtained by density gradient centrifugation, cell phenotype was CD44+CD29+CD105+CD48-CD34-, All surgeries of transplantation for20cases were successful without complications such as renal artery and vein thrombosis, DGF, infections and GVHD.1case in Group control had experienced acute vascular rejection and transplant kidney rupture in the7th day after transplantation, there was no1case of AR occurred in Group experiment. Good graft functions (Cr and Ccr) maintained in Group experiment and Group control.24-hour urinary protein excretion of Group experiment in the early period postoperation (1week) was308.1±55.1mg, lower than that (1727.2±178.1mg) of Group control,with significant difference, P<0.05; there were no statistically difference in lymphocyte count and proportion,the count of T lymphocyte subsets(CD3+CD4+T-cell, CD3+CD8+T-cell) and ratio of CD3+CD4+/CD3+CD8+, Treg proportion between Group experimental and Group control at different observation time points post-operation (1week,1month,3months and6months later), P>0.05,Conclusions:Ideal graft functions can be obtained by autologous bone marrow mesenchymal stem cells combined with low-dose of FK506, it can promote the repair of acute renal injury in the early days. Effective immunosuppressive effect can be induceed by this method, with effective regulation on lymphocyte count and proportion, proportion and ratio of T lymphocyte subsets. It can increase the proportion of Treg cells and induce transplantation tolerance to a certain extent. Objective:To study the effect of BM-MSC to improve renal allograft functions and the mechanisms of BM-MSC to repair chronic renal allograft injury by comparison of parameters about renal functions and renal interstitial fibrosis of the patients with chronic allograft nephropathy before and after the therapy of bone marrow mesenchymal stem cells.Methods:10patients with chronic allograft nephropathy (CAN) were diagnosed by inclusion criteria and enrolled in from2009.12to2012.6.Bone marrow mesenchymal stem cells from third-party healthy donor were prepared and infused three times in total, on day0,day7and1month later. The first infusion of BM-MSC was infused by the pathway of transplant renal artery and guided by DSA, the number of cells was1.0×106/kg,2nd and3rd infusion by the pathway of peripheral vein, cell number5.0×106/kg. Clinical indexes including Cr, BUN, Ccr,24-hour urine protein and blood/urine β2-microglobulin were recorded. Procedural allograft biopsy had been unwent before BM-MSC therapy and6months later, JD image analysis system was applied to detect the changes of interstitial fibrosis indicators of renal allograft including LN, FN,TGF-β1and CTGF.Results:There were no complications such as bleeding, transplant renal artery embolization, pseudoaneurysm and infection,et al,except1case of allergy during the second infusion of BM-MSC.Cr and BUN were203.7±46.4umol/L,15.43±8.46mmol/L before treatment,1week and1month after treatment185.5±46.2umol/L,12.15±4.96mmol/L and172.6umol/L11.76±5.12mmol/L, compared with pre-treatment, the levels of Cr and BUN after therapy had decreased with statistically significant difference (P<0.05). There were no statistical difference between3months and later after therapy and pre-therapy. Ccr of7days,1month and3months after treatmen was43.18±18.04ml/min,43.18in±18.04ml/min,48.04±22.34ml/min respectively, compared with38.01±12.96ml/min pre-treatment, there was statistically significant difference, P<0.05.24hours urinary protein1month after treatment was478.5±289.3mg, less than105.6±327.3mg before treatment,with statistical difference,P<0.05, but without statistical difference in3months and later after treatment and before treatment. The average optical density values of FN and LN,renal interstitial fibrosis markers, were (0.174±0.027,0.199±0.015)6months after treatment,compared with that (0.182±0.019,0.212±0.013) before treatment, the average optical density values had reduced,but without statistical difference,P>0.05; The average optical density values of TGF-β1and CTGF6months after treatment were (0.16±0.015,0.139±0.025), compared with that (0.212±0.017,0.192±0.010) pre-treatment, the average optical density values had reduced with statistical difference, P<0.05.Conclusions:After BM-MSC treatment, serum creatinine(Cr) and urea nitrogen(Bun) of Chronic allograft nephropathy patients can decrease within1month; endogenous creatinine clearance rate(Ccr) can increase within3months;24-hour urinary protein excretion can reduced within1month; the average optical density values of renal interstitial fibrosis markers TGF-β1and CTGF of renal allograft can reduce. |
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