| Objective(s):To analyze the effects of hepatocyte growth factor(HGF)pretreatment bone marrow mesenchymal stem cells(BMSCs)on renal function and renal tissue fibrosis in rats with chronic kidney disease,and to compare the differences between renal artery and tail vein transplantation.Methods:1.Isolation,culture,identification and labeling of BMSCs:two 4-week-old clean grade SD rats,take bilateral femurs and tibias in the ultra-clean worktable.BMSCs were obtained by repeatedly washing the marrow cavity with phosphate buffer saline(PBS),cultured and transferred to the 5th generation.Cell surface antigen was detected by flow cytometry,and differentiation potential of cells was detected by adipogenic and osteogenic induction culture.BMSCs were labeled with EDU in vitro,and the labeling of cells was observed under fluorescence microscope.2.Establishment of CKD model and experimental group:model establishment by repeated injection of ADM into tail vein.81 7-week-old clean grade SD rats,the first dose of ADM was 4mg/kg,two weeks later,the same dose of ADM were injected again.The renal function of the rats was analyzed by detecting the values of serum creatinine(Scr),blood urea nitrogenand(BUN)and the level of 24h of urine protein.Two rats were randomly killed to observe the structural changes of kidneys,confirming that the model rats met the CKD standard.Then the successful rats were randomly divided into 5 groups with 15 rats in each group:①M group(model group);②V-B group:tail vein transplantation BMSCs;③V-HB group:tail vein transplantation BMSCs pretreated by HGF;④A-B group:renal artery transplantation BMSCs;⑤A-HB group:renal artery transplantation BMSCs pretreated by HGF;⑥another 15 healthy rats as normal control group(N group).3.HGF pretreatment BMSCs:BMSCs were co-cultured with HGF at 20μg/L for 24h in pretreatment group,and cultured in low-glucose medium(L-DMEM)in the non-pretreatment group instead of HGF co-culture.The effects of HGF on BMSCs were analyzed by detecting migration and proliferation ability4.Effects of BMSCs transplantation by different ways on renal function and renal tissue fibrosis in rats with CKD:BMSCs transplantation with EDU marker began 10 weeks after the first ADM injection.Group M was injected with PBS by tail vein.BMSCs were transplanted into the tail vein of V-B group.BMSCs pretreated by HGF were transplanted into the tail vein of V-HB group.BMSCs were transplanted by renal artery in group A-B.BMSCs pretreated by HGF in renal artery transplantation group A-HB.Group N was injected with 0.9%sodium chloride solution through tail vein.The above injection volume was 0.5ml,and the number of cells was 3×106cells/ml.At the 2nd,4th and 6th week after stem cell transplantation,the effects of BMSCs on renal function of CKD rats were analyzed by detecting serum creatinine,blood urea nitrogen and 24h urine protein.Five rats in each group were randomly sacrificed and their kidneys were taken for staining.The morphological and structural changes and renal fibrosis of the rats were observed under optical microscope.Fluorescence staining was used to observe the distribution of EDU-labeled BMSCs in different kidney tissues.Immunohistochemical staining was used to compare the expression of transforming growth factor β1(TGF-β1),fibronectin(FN)and alpha smooth muscle actin(α-SMA),and to analyze the effect of BMSCs on renal fibrosis and its possible mechanism.Results:1.The results of BMSCs isolation,culture,identification and labeling showed that the primary BMSCs were transparent round,fusiform after passage,and grew in a fish-like or swirl-like adherent wall,with high proliferative avtivity.CD45 was negative(11.5%)and CD90 was positive(99.6%).Lipogenesis and osteogenesis were induced,differentiated and cultured.In conclusion,the obtained cells were BMSCs,which were successfully labeled with EDU and expressed green fluorescence.2.Establishment of CKD model results:CKD rat model was successfully established 8 weeks after ADM injection.Compared with group N,the serum creatinine,blood urea nitrogen and 24h of urine protein level of M group were significantly increased(P<0.01).HE staining showed a large number of renal interstitial inflammatory cell infiltration with connective tissue hyperplasia,a large number of renal tubules forming protein tubules,glomerular cystectomy dilatation,a large number of glomerular injury,telangiectasia and vacuolar degeneration.Masson staining showed a large area of blue-stained collagen hyperplasia in renal interstitium and hyperplasia around glomerular sac.PASM staining showed marked thickening of the glomerular basement membrane.To sum up,CKD rat model was successfully established.3.HGF pretreated BMSCs:compared with the non-pretreated group,the number of BMSCs in the pretreated group was higher(P<0.05),and the difference in absorbance value at 450nm was not statistically significant(P>0.05).4.Effects of BMSCs transplantation by different ways on renal function and renal tissue fibrosis in rats with CKD:BMSCs transplantation can improve the general condition of rats and increase the body mass gradually.The serum creatinine,blood urea nitrogen and 24h urine protein level of rats in each treatment group were significantly decreased,and the values of serum creatinine,blood urea nitrogen and the level of 24h urine protein in group A-B were significantly lower than those in group V-B in the early stage(P<0.05),while the values of serum creatinine and blood urea nitrogen were still lower than those in group V-B until the 4th week(P<0.05),suggesting BMSCs has positive therapeutic effects for the damage to the kidney,and in a certain period of time,renal artery was superior to the tail vein.There was no significant difference in serum creatinine and blood urea nitrogen between V-B group and V-HB group,A-B group and A-HB group(P>0.05).Only at the second week after transplantation,A-HB group of 24h urine protein levels lower than A-B group,observed at this time,the number of BMSCs in renal tubules and glomerular in A-HB group was more than A-B group,and the number of BMSCs in renal tissue in V-HB group was slightly more than V-B group.It is suggested that HGF pretreatment helps to improve the migration ability of BMSCs,but in the aspect of improving renal function did not show significant treatment effect more than pure BMSCs.The renal tubular interstitial injury score and fibrotic area percentage in the A-B group and the A-HB group were lower than those in the V-B group and the V-HB group(P<0.05),and the expression intensity of TGF-β1,FN and α-SMA was also lower than that in V-B group and V-HB group(P<0.05).However,the differences between the A-B group and the A-HB group,the V-B group and the V-HB group were not statistically significant(P>0.05).Prompted by renal artery BMSCs transplantation effect was more significant in terms of delaying or inhibiting renal fibrosis,and this effect may be related to abate the TGF-β1 mediated renal fibrosis mechanism,although HGF pretreatment could increase the number of BMSCs targeted migrate to the damaged kidney tissues,but to delay the renal fibrosis in this study did not show a remarkable enhancement effect.Conclusion(s):BMSCs have an active repair effect on renal function and can delay or inhibit renal fibrosis.The mechanism is to reduce the excessive deposition of ECM in renal tissue by down-regulating TGF-β1 expression,and the renal artery graft of BMSCs has a better effect than the tail vein in a certain period time.HGF pretreatment can improve the migration ability of BMSCs and increase the number of cells targeting homing to damaged renal tissues,but it does not show significant promotion effect on the improvement of renal function and inhibition of renal fibrosis. |
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