The Hepatocyte-like Differentiation Of HUC-MSCs In Fibrotic And Cirrhotic Rat Model

Liver fibrosis is the repair response of the liver to a variety of pathogenicfactors, such as chronic inflammation, necrosis, or other damage. Cirrhosis isthe end stage of liver fibrosis. At this time, the hepatitis viruses, alcoholmetabolites, bile acids and other liver toxic substances act on the hepatocytes,then liver function significantly decrease, Damaged hepatocytes release theAST, ALT, TBIL, DBIL, reactive oxygen species and fiber medium, whichlead to apoptosis of itself. Currently, liver transplantation is the only effectivemethod for the treatment of end-stage liver disease. However, Donor scarcity,immunological rejection and other reasons limit the clinical application ofliver transplantation. Therefore, it needs to find a new and effective treatmentmethod.Cell therapy is the activity of cell replacement, repairing damaged tissue,thereby enabling the tissues and organs to restore their function. In recentyears, cell therapy has become an effective treatment alternative to organtransplants. The efficacy of stem cell for liver cirrhosis and other end-stageliver disease has been confirmed.Mesenchymal stem cells （MSCs） are a group of stem cells deriving fromMesoderm mesenchymal with multiple differentiatral potential. MSCs can berecovered from a variety of tissues, including bone marrow, umbilical cordtissue, umbilical cord blood, peripheral blood and adipose tissue et al. Undergiven conditions, MSCs can differentiate into bone, nerve cell, hepatocyte andmany other cells. MSCs have many advantages, such as easy to extract, lowimmunogenicity and immunosuppressive effects, which avoid donor scarcityand immunological rejection after liver transplantation. Then MSCs areconsidered to be the most therapeutic potential donor cells. Research hasshown that BM-MSCs can differentiate into functional hepatocytes to repair damaged liver tissue, thus improving liver fibrosis and cirrhosis. Anotherstudy showed that the process of AT-MSCs into hepatocyte in vitro has gonethrough mesenchymal-to-epithelialtransition. Differentiation of MSCs in vivois a dynamic process and whether MET occured in this differentiation processis not clear. For this, we constructed rat liver fibrosis and cirrhosis model byintravenous injection of UC-MSCs to observed the therapeutic effect ofUC-MSCs on liver fibrosis and cirrhosis, the dynamic process ofdifferentiation in vivo and the specific mechanisms of MSCs into functionalhepatocytes, then to provide a theoretical basis for the MSCs treatment of liverfibrosis and cirrhosis.Objective: To research the dynamic process of differentiation of thehUC-MSCs in fibrotic and cirrhotic rat model and its differentiationmechanism.Methods: Male Wistar rats were randomly divided into following groups:control group（CCl₄/saline0wk, n=8）, model group （Liver fibrosis modelgroup: CCl₄/saline4wks,5wks,7wks; liver cirrhosis model group:CCl₄/saline8wks,10wks,14wks, n=8）, MSCs transplantation group （liverfibrosis MSCs transplantation group: CCl₄/MSCs0wk,1wk,2wks,4wks;liver cirrhosis MSCs transplantation group: CCl₄/MSCs0wk,2wks,4wks,8wks, n=8）. The rat model of liver fibrosis and cirrhosis were established byhypodermic injection of CCl₄mixed with olive oil at the concentration of40%（2ml/kg, twice a week）. After the modeling, model group and treatment groupcontinued hypodermic injection of CCl₄twice weekly, until executed. Thetreatment group of liver fibrosis and cirrhosis accept the intravenous injectionof the UC-MSCs （5×106cells）, after the model were successful （4weeks and7weeks respectively）, the rats were killed after MSCs injection at1,2,4weeksand2,4,8weeks. Serological tests were used to detect the ALT and AST,ALB, TBIL and DBIL in rats and to observe the effect of UC-MSCstransplantation on rat liver function; Haematoxylin and eosin staining （H&Estaining）, Masson’s trichrome staining （MT staining） and sirius red stainingwere used to detect liver histopathological changes; The expression of ALB and AFP in liver tissues were measured by immunofluorescence staining andreal-time Q-PCR respectively. The expression of CK18, CK19, vimentin,α-catenin and E-cadherin in liver tissues were observed byimmunohistochemistry and real-time Q-PCR respectively.Results:（1） H&E, MT staining and sirius red staining showed that: withthe administratral time prolonged, degeneration and necrosis of thehepatocytes and deposition of the intrahepatic fibrous tissue graduallyincreased in CCl₄/saline group; compared with that in CCl₄/saline group, thedegeneration and necrosis of hepatocytes decreased in CCl₄/MSCs group. Theinflammatory score and the quantitative analysis of fibrous tissue showedsignificant difference between the above two groups.（2） Serological testshowed that: in liver fibrosis model group, compared with that in CCl₄/saline4wks group, liver function in CCl₄/MSCs1wk has no significant improvement.In CCl₄/MSCs2wks, ALT, AST has no significant differences compared withthat in CCl₄/saline5wks group. In liver cirrhosis model group, compared withthat in CCl₄/saline2wks group, ALB has no significant increase inCCl₄/MSCs4wks group. In the rest CCl₄/MSCs groups, liver function weresignificantly improved at each time point than that in CCl₄/saline group.（3）Immunofluorescence and immunohistochemistry showed that: with the time oftransplantation extended, the expression of ALB, AFP, CK18, CK19,E-cadherin and α-catenin gradually increased, while the content of vimentingradually decreased in the liver fibrosis treatment group; with the extension ofMSCs transplantation time, the expression of ALB and CK18were graduallyincreased, The content of vimentin droped gradually in the liver cirrhosisgroup. Compared with that in CCl₄/saline2wks group, the expression of AFP,CK19, E-cadherin and α-catenin has increased in CCl₄/saline2wks group, thecontent of the above dropped in8weeks after MSCs transplantation than thatin the4weeks, the above several protein were no expression before MSCstransplantation.（4） Western blot analysis suggested that in the liver fibrosisgroup, there were no significant differences between CCl₄/MSCs2wks groupand CCl₄/MSCs1wk group in the level of ALB and CK18protein expression, the expression of ALB and CK18protein significantly increased inCCl₄/MSCs4wks group than that in CCl₄/MSCs2wks group. With theextension of transplantation time, the expression of AFP and CK19proteinwere significantly elevated. In the liver cirrhosis group, the expression of AFPand CK19protein significantly increased in CCl₄/MSCs4wks groupcompared with that in the CCl₄/MSCs2wk group; however, the content ofAFP and CK19protein significantly decreased in CCl₄/MSCs8wks groupthan that in the CCl₄/MSCs4wks group; with the time of MSCstransplantation extended, the expression of ALB and CK18increased, Theabove protein were no expression before MSCs transplantation.（5） Real timeQ-PCR suggested that in the liver fibrosis group, there were no significantdifferences between CCl₄/MSCs2wks group and CCl₄/MSCs1wk group inthe level of ALB and CK18mRNA expression, while the expression of ALBand CK18mRNA significantly increased in CCl₄/MSCs4wks group than thatin CCl₄/MSCs2wks group. With the extension of transplantation time, theexpression levels of AFP and CK19mRNA were significantly elevated. In theliver cirrhosis group, the expression levels of AFP and CK19mRNA weresignificantly elevated in CCl₄/MSCs4wks group compared with that in theCCl₄/MSCs2wks group, however, the content of AFP and CK19mRNAsignificantly decreased in CCl₄/MSCs8wks group than that in the CCl₄/MSCs4wks group; with the time of MSCs transplantation extended, the expressionof ALB and CK18increased, the above mRNA were no expression beforeMSCs transplantation.（6） Western blot analysis suggested that in the liverfibrosis group, with the extension of MSCs transplantation time, theexpression of N-cadherin and vimentin protein gradually decreased; while, thelevels of E-cadherin and α-catenin mRNA expression gradually increased; inthe liver cirrhosis group, compared with that of the CCl₄/MSCs2wks group,the content of E-cadherin and α-catenin protein significantly increasd inCCl₄/MSCs2wks group; the expression of E-cadherin and α-catenin proteinwere significantly decreased in CCl₄/MSCs8wks group than that in theCCl₄/MSCs4wks group, with the time of MSCs transplantation extended, the expression of N-cadherin and vimentin protein were significantly decreased,the above several protein were no expression before MSCs transplantation.（7）In the liver fibrosis group, with the extension of MSCs transplantation time,the expression of N-cadherin and vimentin mRNA gradually decreased; while,the levels of E-cadherin and α-catenin mRNA expression gradually increased;in the liver cirrhosis group, compared with that in the CCl₄/MSCs2wks group,the content of E-cadherin and α-catenin mRNA significantly increasd inCCl₄/MSCs4wks group, the expression of E-cadherin and α-catenin mRNAwere significantly decreased in CCl₄/MSCs8wks group than that in theCCl₄/MSCs4wks group, with the time of transplantation extended, theexpression of N-cadherin and vimentin mRNA were significantly decreased,the above several mRNA were no expression before MSCs transplantation.Conclusions: hUC-MSCs have a therapeutic effect on liver fibrosis andcirrhosis. It is one of the therapeutic mechanism that hUC-MSCs differentiateinto hepatocyte like cell; In vivo, hUC-MSCs into hepatocyte is a dynamicprocess and in this differentiation process, MET occurred.