| Objects1.Cultured、purified and labeled SD rats bone marrow mesenchymal stemcells(BMSCs),used for subsequent therapy by allogeneic infusion.2.Established adriamycin nephropathy rat models similar to human chronic renal disease.3.By maked BMSCs were transplanted in different ways,and the optimal pathway was analyzed by observing renal function improvement and distribution BMSCs in kidney tissues.Then it can be provide reference for clinical treatment of chronic renal diseases.Methods:1.Selected two male SD rats with 12 weeks old,and used to be bone marrow donor.Under aseptic conditions,BMSCs were extracted from the bone marrow cavity when they were executed,then to be separated and purified.It identified as target cells by flow cytometry,subculture to P4-P6,and marked with the Ad5/F35 adenovirus with green fluorescence.2.The model of chronic nephropathy was established by left nephrectomy and three times through the tail vein injected with adriamycin(ADR)(3.0 mg/kg)in SD rats,with 60 males 14-weeks-old CL grade,meanwhile,after 1 week and 2 weeks.Another 12 SD rats were served as normal control group.Biochemical,urinary protein were detected in fourth week after injection of adriamycin.3.48 model rats with nephropathy were randomly divide into 4 groups,and 12 animals in each group:Group A:BMSCs were transfused through the arterial access route,and the same route was used to infuse equal amounts of BMSCs two weeks later;Group V:via the tail Intravenous infusion of BMSCs,two weeks later,injection of equal amounts of BMSCs in the same pathway;IP group:Infusion of BMSCs viacoeliac axis route and infusion of equal amounts of BMSCs in the same pathway two weeks later.The amount of bone marrow mesenchymal stem cells was 2.5 ml per1×106 cells/ml suspension.In the ADR group,no BMSCs were infused.The same amount of physiological saline was infused through the tail vein only when the other groups were infused with BMSCs.12 healthy rats were used as blank group N group.Infusion of BMSCs in other groups was performed via tail vein with 2.5 ml normal saline.After the last transplantation,Hemoglobin,serum creatinine and24h-urinary proteinwere measured at 0 week,1 week,and 2 weeks.At the same time,4 rats in each group(only 3 rats in A group the first two times)were sacrificed and the right renal tissue was taken to observe the distribution of BMCSs.Result:1.The initial bone marrow mesenchymal stem cells showed typical characteristics of adherent growth(24-48 hours),in one week,the cells were round,irregular polygon,only a few showed spindle-shaped.The growth rate of BMSCs was accelerated,and the morphology was regular and long spindle-shaped when passed to the P4 generation.The BMSCs passaged from P4 to P6 were identified by flow cytometry identification,and he positive expression of CD45,CD11b,CD29,and CD44 surface markers were 2.2%,1.9%,97.1%,and 97.9%,respectively.After 6 hours transfected of AD5/F35 adenovirus,the expression of green fluorescence was 75%after changing culture fluid.2.During the modeling process,5 were dead and 3 failed,and only 52 models were successfully established.2 kidneys were sacrificed for renal pathological sectioning.2 were taken as follow-up to the failure of intubating to cause death in Group A.At the 4th week after the last infusion of doxorubicin,the serum creatinine,blood urea nitrogen,and 24-hour urinary protein were elevated in the rats,while serum albumin decreased.Pathological section:The renal morphology showed hardening and atrophy.Part of the glomeruli appeared focal sclerosis,tubular dilatation,renal interstitial inflammation,and fibrosis.Through the above proof.it was demonstrated that the rats models was successful.3.When the first BMSCs were transplanted in group A,1 rat died of postoperative hemorrhage,then one successful model rat was added.One died of the second bone marrow mesenchymal stem cell transplantation and one died after surgery.There were no deaths in the V and IP groups.Serum creatinine,blood urea nitrogen,and 24-hour urinary protein index were significantly higher in group A,group V,group IP,and group ADR at 0,1 and 2 weeks than those in group N(P<0.01).At week 0,there was no significant change in serum creatinine,blood urea nitrogen,24-hour urine protein in group A,group V,and group IP compared with group ADR.At week 1,serum creatinine in group A was significantly lower than that in ADR group(P<0.01),while serum creatinine was not significantly improved in group V and ADR;both 24-hour urinary protein decreased in group A and V were comparable to that in ADR group.and that’s in group A was significantly lower than that in group V(P<0.01).At 2 weeks,the serum creatinine in group A was lower than that in group V and ADR(P<0.01),but there was no significant difference between group V and ADR;the 24-hour urine protein was lower in group A and V than in ADR group(P<0.01),and There was no significant difference between group A and group V.The comparison between the IP group and the ADR group at each time point shows no significant difference among the indicators.Pathology showed focal glomerulosclerosis in the ADR and IP groups,hyperplasia of the basement membrane and mesangial cells,and a large number of inflammatory cells in the interstitium;differences in the glomerular basement membrane and mesangial cells in the V group.Degree of hyperplasia,a small amount of interstitial inflammatory cell infiltration;A group of most glomerular capillary loop expansion,interstitial part of a very small amount of inflammatory cell infiltration;N group pathology showed normal kidney pathology.The pathological results showed that focal glomerular sclerosis,hyperplasia of the basement membrane and mesangial cells in ADR group and IP group,and a large number of inflammatory cells in interstitium;In group V,glomerular basement membrane and mesangial cells showed different degrees of proliferation,and a small amount of interstitial inflammatory cells infiltrated;In group A,most of glomeruli showed angiotelectasis,and a small amount of interstitial inflammatory cells infiltrated.N group pathology showed normal kidney pathology.Detection of green fluorescence showed that:N group,ADR group,and IP group showed no green fluorescence;V group and A group showed green fluorescence on the first day after transplantation,and gradually increased over time,which was the strongest at 14 days;The green fluorescence in group V was mainly expressed in the renal tubules,while group A was not only distributed in the renal tubules,but also in the glomerulus.Conclusion:1.Through cell characteristics detection,the obtained cells were high-purity bone marrow mesenchymal stem cells,which were the target cells required for the experiment,and they were successfully rendered green fluorescent when transfected with adenovirus.2.Unilateral nephrectomy and three infusions of adrimaycin through the tail vein can cause chronic kidney disease in rats.This method is relatively safe,and it is a repeatable,stable,and effective method of modeling.3.Transplantation of allogeneic BMSCs may have different therapeutic effects on rats with chronic kidney disease(CKD)due to different infusion routes:intraperitoneal injection does not improve renal function,and arterial intervention and peripheral intravenous infusion can improve renal function in rats.The effect of the arterial route is better than that of the peripheral vein route.The specific performance of improving renal function is to inhibit blood urea nitrogen and creatinine in rats;increase hemoglobin and improve anemia;reduce urinary protein and increase serum albumin. |
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