Effect Of Human Umbilical Cord Mesenchymal Stem Cells Transfected With HGF On TGF-?1/Smad Signaling Pathway In Rats With Liver Fibrosis

Objective: To explore the possible mechanism of h UCMSCs transfected with HGF in inhibiting liver fibrosis through transplanting h UCMSCs into liver fibrosis rats in vivo,which in order to seek new strategies for the treatment of liver fibrosis.Methods: In vitro,Firstly,h UCMSCs were separated using the tissue block adherence and then divided into three groups,control group: conventionally cultured h UCMSCs;HGF group: h UCMSCs transfected with HGF lentivirus;LV5-NC group: h UCMSCs transfected with lentiviral vector.Observing the cell morphology after transfection at different point in time respectively;the growth curve of each group was delineated through MTT assay;the expression of HGF protein was determined by ELLSA and Western bolt.Secondly,the microcapsules in each group and empty microcapsules(contained no any cells)were prepared by alginate-poly-lysine-alginate(A-P-A)method.In vivo,the rat liver fibrosis model was prepared by the protocol of intraperitoneal injection of CCl4 combined with high-fat low-protein diet for 8w.To verify whether the model was successful,the growth status;the serum biochemical indicators of ALT,AST,ALP,GGT and ALB as well as the pathology of rats in normal control group and CCl4 model group were checked respectively.The successfully model rats were randomly divided into 4 groups: h UCMSCs group,HGF group,LV5-NC group and empty microcapsules group,following by abdominal transplantation of microcapsules respectively.After 2w,the liver pathological changes were determined by HE and Masson staining;the expression of Collagen ?,Collagen ?,TGF-?1,?-SMA,Smad2 and Smad3 protein were determined by immunohistochemistry and Western bolt as well as the level of Collagen ?,Collagen ?,TGF-?1,?-SMA,Smad2 and Smad3 m RNA in each group were checked by RT-PCR.Results: 1.In vitro,there were no significant difference in cell morphology,cell proliferation ability in HGF group and the LV5-NC group compared with control group.The protein expression of HGF in HGF group was significantly higher than LV5-NC group as well as control group(P<0.01),whereas the expression had no significant difference between LV5-NC group and control group(P>0.05).2.In vivo,after 8w of intraperitoneal injection of CCl4 combined with high-fat low-protein diet,the level of ALT,AST,ALP and GGT in CCl4 group were significantly increased compared with normal control group(P<0.05),whereas the level of ALB was significantly decreased(P<0.05).The results of HE and Masson staining suggested that the hepatic lobular structure in normal control group was orderly,radiate,the morphology of the hepatocytes was integral and the structure of the hepatic sinusoid was normal.In addition,there was no inflammatory cell infiltration around the portal area besides few collagen fibers.However,in CCl4 group,the structure of the hepatic lobular was destroyed,with disordered arrangement in hepatocytes,steatosis,inflammatory cells infiltration and significantly increased of collagen fibers.3.After abdominal transplantation of microcapsules for 2w,the results of HE and Masson staining suggested that the degree of hepatocyte necrosis,steatosis,inflammatory cells infiltration and fibrotic lesion were significantly reduced;the expression of Collagen ?,Collagen ?,TGF-?1,?-SMA,Smad2 and Smad3 were significantly decreased both in m RNA and protein level(P<0.05)in HGF group,h UCMSCs and LV5-NC group compared with empty microcapsules group.The fibrosis was significantly alleviated;the m RNA and protein level of Collagen?,Collagen ?,TGF-?1,?-SMA,Smad2 and Smad3 were more significantly decreased(P<0.05)in HGF group compared with h UCMSCs and LV5-NC group,Whereas the indices mentioned above had no significant difference between h UCMSCs and LV5-NC group(P>0.05).Conclusion: 1.h UCMSCs carrying HGF gene could decrease the expression of collagen and ?-SMA.thereby inhibiting liver fibrosis.2.h UCMSCs transfected with HGF could alleviate the expression of TGF-?1,Smad2 and Smad3.Furthermore,the mechanism of prevent liver fibrosis was closely related to the inhabitation of TGF-?1/Smads signaling pathway.