| BackgroudRenal transplantation is regarded as the most effective treatment for end-stage renal failure,however,the postoperative rejection restricts its efficacy currently.Currently,acute allograft rejection is well treated with high dose prednisolone and immunosuppressive agent,but long-term use of immunosuppressants will be detrimental to the long-term survival of the transplanted kidney and will have a lot of side effects on the human body.Thus,a local and alloantigen-specific immunological tolerance may be a promising approach to improve kidney transplantation.Bone Mesenchymal Stem Cells(BMSCs)represent a type of progenitor cells which is characterized by self-renewal and differentiation potential,they can differentiate into a variety of tissues,including adipose,bone,cartilage,liver,kidney and so on,under different induction conditions,also have low immunogenicity and immunosuppressive effect.They preferentially migrate to the injured tissue and promote tissue repair but with limited efficiency.The limited number of transplanted cells to the injured kidney is one of the reasons that affect the repair efficiency.Erythropoietin(EPO),a glycoprotein that regulates the growth and differentiation of erythroid progenitor cells,has been extensively demonstrated for its effectiveness in the treatment of kidney injury.At present,few studies have shown whether EPO is beneficial to the targeted renal homing and paracrine of BMSCs transplantation,thereby enhancing the therapeutic effect of transplantation and reducing the incidence of rejection after renal transplantation.This study preliminarily investigated whether EPO pretreatment could reduce rejection after BMSCs treatment.ObjectivesA rat model of allogeneic renal transplantation was established.Bone marrow mesenchymal stem cells(BMSCs)pretreated with erythropoietin epoxide(EPO)were injected via tail vein to observe the effect of exogenous EPO on preventing rejection after BMSCs transplantation.Method1、Preparation,experimental grouping and treatment of rat kidney transplantation modelSPF-level SD and Wistar rats(24 rats each)were fed for 3 days in the quarantine room.Wistar rats were used as donors,and SD rats were used as recipients to establish the renal transplantation model.After kidney transplantation modeling,the rats were randomly divided into 4 groups(n=6/group).Both donor and recipient rats were anesthetized by intraperitoneal injection with 2%pentobarbital sodium(30 mg/kg).Left donor kidney was harvested and than perfused with 1%heparin water,and after it turned pale,it was preserved at 0-4 ℃.Orthotopic transplantation was performed using a conventional end-to-end anastomosis between blood vessels with interrupted sutures.After recovering the renal blood flow,if it was well perfused within 5 minutes,then an end-to-end anastomosis using 4 interrupted stitches was performed anastomosing the ureter,after that we closed the abdominal cavity.Operation process pictures were taken.The rat model of kidney transplantation was successfully established as follows:for the EPO group,1 ml of 500 IU/ml EPO was immediately injected via the caudal vein after renal transplantation;for the BMSCs group,1 ml of PBS solution containing 1×106 BMSCs was injected via the caudal vein after kidney transplantation;and for the EPO-BMSCs group,1 ml of PBS solution containing 1×106 BMSCs pretreated by EPO was injected into the caudal vein after renal transplantation.All rats were housed at favorable temperatures and humidity with an unlimited supply of water and food and were sacrificed(3 rats at each time)for each group,at 1 d and 5 d after renal transplantation.Blood samples were collected through the inferior vena cava,and the serum was isolated and stored in a refrigerator(-20 ℃).After perfusion with 1%heparin sodium via the aorta,the bilateral kidneys were separated and further perfused with heparin sodium.The corticomedullary junction of the kidney was cut for western blot.After fixing with 10%formalin,the kidney tissue was pathologically analyzed.2、Scr levelOn the 1st and 5th day after renal transplantation,the blood samples were collected from the renal transplantation rats,the serum was isolated,and the serum creatinine was detected with the kit.3、Pathological changesAfter fixation in 10%formalin,the fixed recipients’ renal tissues were used to make paraffin sections.Then,cut them into 5 im thick sections,and stained with hematoxylin and eosin(H&E).Each case was randomly observed and photographed with 10 unoverlapped renal units under(200×)high-power microscope.The possibility of inflammation and rejection was analyzed.4、Western blotThe 80～100 mg of renal tissue samples were treated with 1 ml of lysis buffer and centrifuged at 20,000 rpm(4℃)for 10 min.The supernatant was centrifuged for another 10 min at 12,000 rpm(4℃).The protein concentration was measured by BCA kit,30 μg protein was used to do the SDS-PAGE electrophoresis.Protein was transfered to PVDF membrane,and then PVDF membrane was shaked at 70rmp for 1h in 4 mL solution containing 5%BSA/TBST at room temperature,adding 4 mL antibody/BSA dilution and incubated at room temperature for half an hour,and next shaked in refrigerator incubation at 4 ℃ overnight.TBST washed membrane,70rmp 3 times,per 10 min with horseradish peroxidase labeled Goat anti rabbit IgG(1:2000)as second antibody,shaked for 1h at room temperature,washing the membrane 3 times/10 min in chemiluminescence imaging system(GelDoc XR+),and images was automatically acquired by CCD.β-actin was used as the internal reference and the protein expression was the ratio of the two band gray values.The protein bands were concluded,respectively,by ImageJ image software.5、Statistical analysisThe results were expressed as the means±standard deviation.Student’s t-test was performed to analyze the differences between the two groups.Multiple-group comparison was performed using one-way analysis of variance(ANOVA)followed by a Student-Newman-Keuls test.SPSS 19.0 statistical software was used for the analysis.Values of P<0.05 were considered statistically significant.Results1、Scr levelAt the first day after modeling,compared with the EPO-BMSCs group,the Scr levels in the control group,EPO group and BMSCs group were higher to some extent.However,the differences were not statistically significant(P>0.05).At the fifth day after operation,compared with the EPO-BMSCs group,the Scr levels in the control group,EPO group and BMSCs group were markedly higher,and the differences are statistically significant(P<0.05).2、Pathological changesAt the first day after transplantation,inflammation occurred in every group and acute rejection occurred in two of three recipients in the control group,and no acute rejection occurred in the other groups.Every transplanted kidney was swollen in shape.At the fifth day after operation,in the control group and EPO group,acute rejection occurred in all three recipients each group.Acute rejection occurred in one of three recipients in BMSCs group.There was no acute rejection in EPO+BMSCs group.Every transplanted kidney was swollen in shape and the occurrence of acute rejection of the transplanted kidneys were dark red in colour.3、Western BlotWestern blot analysis showed that at the first day after modeling,compared with the EPO-BMSCs group,the ratios of gray values for IFN-γ in the control group,EPO group and BMSCs group were higher.However,the differences between the EPO-BMSCs group and BMSCs group were not statistically significant(P>0.05).At the fifth day,compared with the EPO-BMSCs group,the ratios of gray values for IFN-y in the control group,EPO group and BMSCs group were clearly higher,and the differences were statistically significant(P<0.05).At the first day after transplantation,compared with the EPO-BMSCs group,the ratios of gray values for IL-4 in the control group,EPO group and BMSCs group were higher.However,the differences were not statistically significant(P>0.05).At the fifth day,compared with the EPO-BMSCs group,the ratios of gray values for IL-4 in the control group,EPO group and BMSCs group were different,but the differences were not statistically significant(P>0.05).At the first day after operation,compared with the EPO-BMSCs group,the ratios of(IFN-γ/IL-4)in the control group,EPO group and BMSCs group were higher.However,the differences between the EPO-BMSCs group and BMSCs group were not statistically significant(P>0.05).At the fifth day,compared with the EPO-BMSCs group,the ratios of(IFN-γ/IL-4)in the control group,EPO group and BMSCs group were distinctly higher,and the differences were statistically significant(P<0.05).Conclusion:The transplantation of BMSCs pretreatment with EPO may be an effective way to prevent acute rejection after renal transplantation,which may increase the number of injected mesenchymal stem cells to the transplanted kidney and regulate the expression of related cytokine,such as the expression of IL-4,IFN-γand IFN-γ/IL-4 to regulate the balance of Th1/Th2,and improve the microenvironment of renal transplantation to prevent the occurrence of rejection. |
PDF Full Text Request