Human Umbilical Cord Mesenchymal Stem Cells Slow Down The Fibrosis Induced By Adriamycin In FSGS Mice And Its Related Mechanisms

Objective To investigate the protective effect of human umbilical cord mesenchymal stem cells(HU-MSCs)cultured,identified and labeled in vitro on adriamycin-induced FSGS mice and the related mechanism.Method(1)Under the sterile conditions,the human umbilical cord was taken,and the HU-MSCs were obtained by enzymatic hydrolysis.The obtained primary stem cells were screened and subcultured to observe the cell morphology;the cell surface antigen of HU-MSCs was detected by flow cytometry;Standby;labeled PKH-26 after resuscitation.(2)36 8-week-old male Balb/c mice were randomly divided into two groups after one week of adaptive feeding:experimental group(n=24),one-time intravenous injection of doxorubicin at 10.5 mg/kg body weight;Group(n=12),the same method was used to inject an equal amount of0.9% sterile saline.On the 7th,14 th and 28 th days,the body weight and urine were measured respectively,and the body weight of the mice was observed and the urinary albumin content of the mice was detected.All the mice were subjected to eyelid blood sampling for 28 days,and serum creatinine and urea nitrogen were detected.Six rats were killed by light microscopy to determine whether the model preparation was successful.(3)After successful modeling,18 mice in the experimental group FSGS model were randomly divided into FSGS model group(FSGS,n=6),stem cell treatment group(FSGS+HU-MSCs,n=6),saline treatment group.(FSGS + NS,n = 6),6 normal mice were used as a control group(Control).In the FSGS+HU-MSCs group,HU-MSCs(2*106)were injected into the tail vein on the 33 rd,40th,47 th,54th and 61 st day respectively.The FSGS+NS group and the Control group were injected with the same amount of 0.9% at the same time.Sterile saline was used as a control.Body weight and24 h urine were collected in each group on the 54 th and 68 th day of the experiment.The body weight of the mice was observed again and the urinary albumin content of the mice was detected.All mice were sacrificed on the 68 th day of the experiment.Blood and kidney samples were collected,serum creatinine and urea nitrogen were detected.The pathological changes of renal tissues were observed by HE,PAS and Masson staining.TGF-β1 and Smad3 were detected by immunofluorescence semi-quantitative method.Smad7 protein expression.Results(1)Culture and identification results of HU-MSCs:The cells isolated by collagenase digestion began to adhere after 24-48 hours,and the adherent cells were mainly spindle-shaped after 3-5 days,after 10-14 days.The fusion rate after cultivation can reach 80-90%.Flow cytometry results showed that HU-MSCs specifically expressed epithelial cell surface antigens such as CD29,CD44,CD90,CD105,CD126,but not hematopoietic cells,endothelial cell surface markers such as CD34,CD45 and CD31,and did not express major tissue phases.Capacitive antigen HLA-DR.(2)The results of doxorubicin-induced FSGS model showed that the body weight of the experimental group was significantly lower than that of the control group(P<0.01),and the levels of urinary albumin,serum creatinine and urea nitrogen were significantly higher than those of the control group(P<0.05).The 28-day renal pathology showed mild hyperplasia of mesangial cells and mesangial matrix,and the segment was moderately aggravated.Some of the capillary stenosis was compressed,balloon adhesion was associated with podocyte proliferation,and tubular epithelial cells were swollen and degenerated.Tube type,lymphatic infiltration of renal interstitial,thickening of small vessel wall.(3)Results after HU-MSCs transplantation were shown: FSGS+HU-MSCs group,FSGS group,FSGS+NS group and control group showed significant weight loss(p<0.001),urinary albumin,serum creatinine,urea Nitrogen levels increased significantly(p<0.001);FSGS+HU-MSCs group had significantly higher body weight than FSGS group and FSGS+NS group(P<0.05),and urinary albumin,serum creatinine and urea nitrogen levels decreased significantly(P<0.05).<0.01);On the 68 th day of the experiment(the 35 th day after stem cell treatment),renal pathology showed mild proliferation of mesangial cells and mesangial matrix,disappearance of renal tubular type,significant reduction of vacuolar degeneration,and renal interstitial lymphoid Cell infiltration and fibrosis were alleviated,and no hyperemia occurred.Immunohistochemistry showed that the expression of TGF-β1 and Smad3 was significantly increased in FSGS group and FSGS+HU-MSCs group compared with Control group(P<0.001),and Smad7 expression was significantly decreased(P< 0.001);while in the FSGS+HU-MSCs group,the expression of TGF-β1 and Smad3 was significantly decreased compared with the FSGS group,and the Smad7 protein level was significantly increased,and the difference was P<0.001.Conclusions HU-MSCs may repair renal fibrosis injury in FSGS mice through TGF-β1/Smad signaling pathway and slow the progression of the disease.