CM-Dil-labeled Human Umbilical Cord Mesenchymal Stem Cells Produce Therapeutic Function On Acute Liver Failure Rats

Background:Acute liver failure,as a clinical syndrome,is characterized by jaundice,ascites,hepatic encephalopathy,and a bleeding tendency due to impairment of liver function.The liver transplantation is considered as the first-line therapy for acute liver failure,but it is limited to the shortage of liver donors and the high cost of operation.Mesenchymal stem cells(MSCs)are proposed as a promising therapy for the liver failure,but the homing ability of MSCs into the liver occurred in just a limited number.The previous studies showed that the concentration of hepatocyte growth factor(HGF)in the injured liver was significantly higher,and the c-Met modified bone marrow mesenchymal stem cells targeted homing to injured liver.However,the number of MSC derived bone marrow is limited,and their biological characteristics are influenced by aging.In recent years,more and more attention has been paid to MSCs derived human umbilical cord.Compared with MSCs derived bone marrow,MSCs derived umbilical cord are superior in proliferation and differentiation.During expansion,with a higher number of passage,there is a decrease in efficacy of homing.Objective:To invesigate the effect of CM-Dil labeled human umbilical cord MSCs with different passage on liver failure,and the underlying mechanism of homing among different passage human umbilical cord MSCs.Methods:1.36 male SD rats,weighing 200-230 g,were randomly assigned into 3 groups(P5treatment group,P10 treatment group and control group,n=12).The co-injection of D-galactosamine(D-Gal N,1000 mg/kg)and lipopolysaccharide(LPS,10 ?g/kg)were injected intraperitoneally.24 hours later,the transfusionof the P5 h UC-MSCs and P10 h UC-MSCs(1.0×107 cells/kg/ml)were transplanted via the vena caudalis respectively,while1 ml of normal saline were tranfused into the control group.The survival rate of three groups was also observed daily.Meanwhile,the blood samples from the internal hemorrhoid veins were collected from the three groups at 0,24,48,and 72 hours for liver function tests.At the same time,hepatic tissue specimens were collected at 24,48,and 72 hours after the rats were modeled for HE staining and immunohistochemical staining.2.The CM-Dil labeled P5 and P10 hUC-MSCs(1 x 106 cells)were infused into the acute liver failure rats through the tail vein.After 24 h,two groups of rats were killed,then the homing efficacy were observed by fluorescence microscope,and analyzed by Image-Pro plus.3.The expression of PCNA and the apoptosis of TUNEL cells were determined by immunohistochemistry staining,and the data was analyzed by Image Pro Plus.4.Flow cytometry was used to detect the cell cycle of P5 and P10 generation h UC-MSCs;Western blot was used to detect the expression level of c-Met protein in the P5 and P10 generation of h UC-MSCs.Results:1.The survival rate of P5/P10/control group was 62.5%?37.5%?0%50% respectively.To compare the liver function and HAI scores of the P5 h UC-MSCs to those of theP10 h UC-MSCs,improved liver functionand decreased HAI scores in the P5hUC-MSCs were better than in the P10 hUC-MSCs.2.The number of homing cells in the P5 h UC-MSCs treatment group was significantly higher than that in the P10 h UC-MSCs treatment group via fluorescence microscopy.3.The immunohistochemical staining of liver tissue found that,compared to the P10 h UC-MSCs group and the control group,in the P5 h UC-MSCs group increased and decreased significantly the IOD of PCNA and TUNEL respectively.4.The cycles of the P3,P5,P8 and P10 hUC-MSCs respectively detected by flow cytometry(71.3%,60.1%,37.4% and 29.9% respectively),suggesting that a higher number of passage there is a decrease in proliferation.c-Met expression in P3,P5,P8 and P10 h UC-MSCs detected by Western-blot indicates that a higher number of passage there is a decrease in c-Met.Conclusion:1.Transplantation of hUC-MSCs could improve rat survival rate and improve liver function in rats,and there are differences between different generations.2.The therapeutic effect of hUC-MSCs on liver failure may be related to promoting proliferation and inhibiting apoptosis of hepatocytes,and there are also differences between different generations.3.The differences in the therapeutic effects of different generations of h UC-MSCs may be related to the cell homing effect,and the mechanism may be related to the c-Met expression levels of different generations of h UC-MSCs.