| Objective:1.To investigate the effects of umbilical cord mesenchymal stem cells(UC-MSC)from human on the liver and kidney injury induced by cyclophosphamide(CTX);2.To explore the effects of canonical Wnt/β-catenin signaling pathway on UC-MSC protecting the kidney injury induce by CTX.Methods:1.The isolation,culture and identification of umbilical cord mesenchymal stem cells(UC-MSCs).The cells were isolated from fresh umbilical card and cultured to passage 4 to observe the cellular morphology.The cytophenotypic markers were identification by flow cytometer(FCM).The cell differentiation to osteocyte,chondrocyte and adipocyte was induced by special medium.2.The building and identification of the model of liver and kidney injury induced by cyclophosphamide.The SD rats were divided randomly into Control group and CTX group.The rats in CTX group were injected intraperitoneally CTX(200mg/kg)and the Control group was injected PBS at the same way.All of the rats were sacrificed after 24h of injection.2.1 The detections of biochemical index in serum The peripheral blood was centrifuged(3500g,20min)to obtain serum.The alanine aminotransferase(ALT),aspartate aminotransferase(AST),alkaline phosphatase(ALP),total bilirubin(TBIL),blood urea nitrogen(BUN)and creatinine were detected in serum.2.2 The detection of oxidative stress-injury index in the homogeneity of liver and kidney The 10%liver and kidney tissue homogenate were extracted by ultrasonic method.The malondialdehyde(MDA),lipid peroxide(LPO),nitric oxide(NO)glutathione(GSH),glutathione peroxidase(GSH-PX),and total superoxide dismutase(T-SOD)were detected in the tissue homogenate.2.3 The histopathological examination of liver and kidney The tissues were pieced and straining with hematoxylin and eosin(H&E).3.The effect of UC-MSCs on the injury of liver and kidney induced by CTX in ratsThe male SD rats were divided randomly into Control group,CTX group and CTX+UC-MSC group.The day when the UC-MSCs were injected in the CTX+UC-MSC group on the first time was the first day(1st).The rats were injected with UC-MSC(2×10^6)。PBS via the tail vein at the 1st,4th,7th,10th day while CTX group and CTX+UC-MSC group were intraperitoneally injected CTX(200mg/kg)at the 3rd day.The rats selected randomly from 3 groups were sacrificed at the given time.3.1 The detection of ALT,AST,ALP,TBIL,BUN and creatinine in the serum of different groups by chemical analysis.3.2 The detection of MDA,LPO,NO,T-SOD,GSH and GSH-PX of the tissues homogeneity of kidney and liver by biochemical methods.3.3 The mRNA expression of apoptosis-associated protein and vascular endothelial growth factor(VEGFA)in the liver and kidney tissue.The total mRNA of liver and kidney was extracted and detected the gene expression of apoptosis-associated protein(Bax and Bcl-2)and VEGFA.3.4 Pathological examination of liver and kidney at different groups The tissue were pieced and strained with H&E and immunohistochemistry(a-SMA and Ki-67 straining).4.The effects of Wnt/b-catenin signaling pathway on the UC-MSC treating the kidney injury induced by CTX.The total protein was extracted from the kidney tissues and detected the protein expression of Wnt3,β-catenin,Axin2,occludin,a-SMA and VEGFR1 by western blot.Results1.UC-MSCs were isolated and cultured successfully The cultured cells showed a spindle-shaped morpholopy and formed a monolayer of typical fibroblastic cells,which were positive expression for CD44,CD73,CD90 and CD105,and negative expression for CD34 and CD45.The adherent cells could be induced to differentiate into osteocyte,chondrocyte and adipocyte.All of the performance above testified the spindle-shaped cells were the UC-MSC.2.The model of liver and kidney injury induced by cyclophosphamide was established successfully.2.1 The expressions of ALT,AST,ALP and TBIL in serum wereelevated compared with the Control group,which suggested the liver function was damaged.The expressions of BUN and creatinine were up-regulated in serum compared with the Control group,which mean the kidney function was injury(P<0.05).2.2 Compared with the control group,the expressions of MDA,LPO and NO in the liver and kidney tissues homogeneity of CTX group were rise and the expressions of GSH,GSH-PX and T-SOD were on the contrary,which suggested the CTX intraperitoneally could induce the oxidative-stress injury of liver and kidney(P<0.05).2.3 The hepatocellular damage could be observed clearly in the CTX group with significantly edema and vacuolization,a good deal of inflammatory cells compared with the Control group.The histopathology behavior in CTX-induced kidney injury revealed moderate inflammatory infiltrate,necrosis and atrophy inglomeruli and swelling with vacuolization.3.UC-MSC injected via the tail vein could alleviate the liver and kidney injury induced by CTX.3.1 Compared with the CTX group,the content of ALT,AST,ALP,TBIL,BUN and creatinine in the serum of rats in the CTX+UC-MSC were declined at any point in time(P<0.05).3.2 The expression of SOD,GSH and GSH-PX were more in the homogeneity of liver and kidney were more in the CTX+UC-MSC group than the CTX group(P<0.05),whereas the expression of MDA,LPO and NO were on the contrary(P<0.05).3.3 The real-time qPCR showed the mRNA expression of Bax in the CTX group was elevated and the Bcl-2 was declined.The level of Bax after treatment of UC-MSC via tail vein was less than the CTX group and the level of Bcl-2 was on the contrary.The mRNA content of VEGFA in the liver and kidney of CTX+UC-MSC group was significantly more than the Control group and the CTX group.3.4 The effects of UC-MSC on histopathologic changes in CTX-induced kidney tissuesThe hepatocellular injury could be observed obviously in the CTX group with marked edema and vacuolization,a mass of inflammatory cells compared with the Control group.In the CTX+UC-MSC group,the extent and area of congestion,the infiltration of inflammatory cells and cell death were attenuated.The histopathology condition in CTX-induced kidney revealed moderate inflammatory infiltrate,necrosis and atrophy in the corpuscle and swelling with vacuolization of the proximal convoluted tubules cells.However treatment with UC-MSC effectively moderated the severity of renal condition.3.5 Immunohistochemical straining There was no a-SMA+ cells in the liver of Control group.The expression of α-SMA + cells in the liver at D4 was diffused distribution and there were a good deal of positive cells at D13.The expression in the CTX+UC-MSC group was less than the CTX group at any point in time.The content of Ki-67 was focused on the inner surface of the liver blood vessels in the Control group,whereas the Ki-67+cells were diffused distribution in the liver parenchyma.The expression of Ki-67+cells was more in the CTX+UC-MSC group than the CTX group at any points in time,which were focused on the liver parenchyma and perivascular.There was no a-SMA + cells in the kidney of Control group.After the injection of CTX,a-SMA + cells were expressed highly in the glomeruli of kidney at D10 and D13,and the content was less in the CTX+UC-MSC group than the CTX group.In the control group,the Ki-67+cells were concentrated around the renal tubule.And the positive cells were more after the injection of UC-MSC than the CTX group.4.The expression of Wnt3,β-catenin and Axin2 in the CTX+UC-MSC group,which were the key protein of Wnt/β-catenin signaling pathway,were elevated and the occludin and VEGFR1 expression was increased.The content of a-SMA protein was decreased.Conclusions:1.The pre-treatment of UC-MSCs can reduce the liver and kidney injury induced by CTX,the injection of UC-MSC multiply via tail vein can accelerate the recovery of liver and kidney;2.The mechanism of the treatment may be the activation of canonical Wnt/β-catenin signaling pathway. |
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