Saldroside Can Differentiate Bone Marrow Derived Mesenchymal Stem Cells Towards Hepatocytes And Reverse Autoimmune Liver Fibrosis

Chapter one:IntroductionThe liver is the largest digestive gland in the body and it has a multitude of important and complex functions,such as synthesizing proteins,synthesizing,storing, and metabolizing fats,metabolizing and storing carbohydrates,forming and secreting bile,regulation of the endocrine,blood coagulation,as well as participating in the immune system,eliminating the potentially harmful biochemical products produced by the body,and detoxifying.Once liver is de-function or function failure,serious threat to human health may be caused.Therefore,liver diseases are the main causes of death in the world.The data published by the World Health Statistics in 2005 showed that the total quantity of hepatitis B virus(HBV) in the world has infected more than two billion people,of which 350 million more will become chronic hepatitis B patients. Approximately 100 million people each year die from liver failure,liver cirrhosis, hepatocellular carcinoma and infections caused by hepatitis B.According to the survey in 2008,about 7.18 percent of the total population are hepatitis B surface antigen(HBsAg) positive,and there are 93 million hepatitis B surface antigen carriers in China,while more than 30 million patients suffered from HBV-induced liver fibrosis and cirrhosis.Therefore substantial financial and material resources are required to pay for treatment every year,and the state and individuals bear this heavy financial burden.From the treatment point of view,there are many methods to treat liver diseases, such as treatment according to a cause of disease(such as anti-virus),symptomatic treatment(e.g.anti-fibrosis and immune regulation),liver and nutritional support,gene therapy,liver transplantation,liver support treatment and cells(liver cells,or stem cell) transplantation and so on.However,liver transplantation is currently ultimate method for the clinical treatment of liver disease,while a transplant operation is getting more advanced,and saves many patients of liver failure.However,due to donor source,graft versus host reaction,costly surgery,and many other factors,liver transplantation in clinical application is restricted.Liver cell transplantation is an advanced therapy,which can partially overcome the problems of liver transplantation,and has the following advantages:simple operation,minimally invasion,low-cost;abundant source,a donor liver for more patients;cells can be frozen,will not result in a shortage and the weak graft versus host reaction.However,liver cell transplantation has not been applied in clinical practice because many problems are not yet resolved.Bottlenecks of liver cell transplantation are cell supply and immune rejection.Presently allogeneic mature liver cells are used for clinical application,but it is difficult to obtain many mature cells and to amplificate in vitro because of the loss of cross-talking in cells;what is more,liver cells transplanted may gradually apoptosis and fail to produce the desired therapeutic effect due to immune rejection.How to solve these questions? Now fetal liver cells and embryonic stem cells are ruled out of the clinical application due to medical ethics,and then we applied to stem cells of one's own because autologous cells cannot cause immune rejection and stem cells proliferate faster.It has recently been reported bone marrow derived mesenchymal stem cells(BM-MSCs or MSCs),one of adult stem cells,can differentiate towards liver cells in vitro and in vivo.As a rich source of stem cells,an application of autologous MSCs for liver disease is very promising.Taking an overview of the studies of MSCs presently,both basic research and pre-clinical trials have not yet had outstanding results. Separation,identification,and differentiation of MSCs,maintenance of character, biological security and comprehensive evaluation of efficacy are required the in-depth studies.Goals of our group are to explore the mechanism and controllable conditions of differentiation of MSCs,to research new drugs of induced differentiation;to verify the therapeutic efficacy and mechanism of MSCs in the treatment of autoimmune liver fibrosis.We wish MSCs transplantation could apply in clinic,a simultaneous development of stem cells and Chinese medicine-related industries,which will open up new economic growth point.Chapter two:Isolation,cultivation and CharacterizationAim:To optimize the isolation,cultivation and amplification in vitro and to explore biological characteristics and differentiation potential of rMSCs.Methods:Under sterile conditions,Sprague Dawley(SD) rats(one month old) rats were sacrificed,bone marrow cells were collected,suspended in the NH₄Cl hypotonic salt solution(erythrocyte lysis buffer) for erythrocyte lysis,and centrifuged to collect the cells for cultivating.Low glucose DMEM(LG-DMEM),supplemented with 10% fetal bovine serum(FBS) was applied.When the culture reached 80%confluence,it was pre-incubated with collagenase for 1～2 hours,cells were trypsinized and a serial subcultivation began.To determine growth characteristics of the cells,their morphology was observed under the inverted microscope and the cells growth curve was made.To determine the antigen expression profiles of the cells,surface antigens such as CD45, cluster of differentiation(CD) 44,CD90,were analyzed using a FACScan flow cytometer.To verify the multipotential mesenchymal characteristics,cells of passage 3 were analyzed for adipogenic,chondrogenic and osteogenic differentiation.Result:Without interference from erythrocytes,the adherent spindle-shaped cells were observed in 4-6h.Cells rapidly grew colonies on day 4-6 and the culture reached 80%confluence 7-8 days later.Primary cells and passage cells both exhibited homogeneous fibroblast-like morphology and grew as a whirlpool.Passage cells proliferated quickly on day 3 and reached a plateau of confluence on day 5-6.They were subcultured for more than 20 times stably.Flow cytometry analyses revealed that the adherent fibroblastic cells of passage 3 were negative for CD45 while they were positive for CD44 and CD90,known as cell surface antigens of rMSCs.Differentiation assays of rMSCs revealed that the cultured cells could differentiate into osteoblastic, chondrogenic and adipocytic lineage and have the corresponding characteristics when cultured in osteoblastic,chondrogenic and adipocytic induction medium,respectively.Conclusion:The more pure rMSCs could be isolated by lysing erythrocytes associated with adherent culture system.The adherent cells were subcultured in low glucose-Dulbecco's Minimum Essential Medium(DMEM) supplemented with 10% FBS for more than 20 times and their characters were stable.Immunophenotypic analysis revealed expression of CD44 and CD90 and no expression of CD45,which belongs to hematopoietic cell surface antigens.Differentiation assays of rMSCs revealed that the cultured cells could differentiate into osteoblastic,chondrogenic and adipocytic lineage and have the corresponding characteristics when cultured in osteoblastic,chondrogenic and adipocytic induction medium,respectively.Therefore, we adopted this method to isolate the cells is exactly bone marrow-derived mesenchymal stem cells.It is easy to obtain the pure rMSCs by lyzing erythrocyte with hypotonic salt solution in combination with adherent culture system.The method will make isolation,cultivation and amplification easy.Chapter three:Salidroside differentiates rat MSCs towards hepatocytes in vitroAim:To establish and optimize the special induction system of differentiating rMSCs towards hepatocytes in vitro;to screen small-molecule compounds which come from traditional Chinese medicine and can induce rMSCs to differentiate towards hepatocytes;to evaluate the hepatocytes of hepatic differentiation and to underlay the follow-up experiments,induction of differentiation from comprehensive evaluation of the liver cells,in order to lay the foundation for and to bridge findings in vitro and application in vivo.Methods:(1) salidroside in association with fibroblast growth factor(FGF)-4 could differentiate rMSCs towards hepatocytes,rMSCs of passage 3 were incubated in basic differentiation DMEM medium(high glycose),supplemented with 2%FBS.When reaching confluence,rMSCs were serum deprived for 2 days,in DMEM supplemented with FGF4,prior to induction.Differentiation was induced by treating rMSCs with differentiation DMEM medium,rMSCs were divided into three groups according to supplement:Group A(negative control),B(positive control) and C were supplemented with FGF4,FGF 4 & hepatocyte growth factor(HGF),and FGF 4 & salidroside, respectively.Cell morphology was observed under inverted microscope every day after the induction,rMSCs,induced for day 0,7,14 and 21 days,were harvested to analyze the typical hepatic characteristics,such as gene expression of HGF,hepatocyte growth factor receptor(HGFR),hepatocyte nuclear factors(HNF)-3β,cytochrome P450 (CYP)2B1 & 1A1,alpha fetoprotein(AFP),Albumin(ALB),cytokeratin(CK)18 and transthyretin(TTR),protein expression of AFP,ALB and CK18,CYP450 (CYP450)-dependent activity and inducibility,cellular uptake of low density lipoprotein (LDL) and urea synthesis.(2) salidroside could alone differentiate rMSCs towards hepatocytes.Methods of incubated and pre-induced were carried out as described above. rMSCs were divided into several groups according to supplement:Group A(negative control),B(positive control),C,D and E were supplemented with FGF4,FGF 4 & HGF, 10μM salidroside,20μM salidroside and 50μM salidroside,respectively,rMSCs, induced for day 0,7,14,were harvested to analyze the expression of AFP and ALB, which are hepatic proteins.(3) The mechanism by which salidroside differentiates rMSCs towards hepatocytes.Methods of incubated and pre-induced were carried out as described above,rMSCs were divided into several groups according to supplement: Group A(positive control),B(negative control),C,D,E and F were supplemented with 20μM salidroside and salidroside & LY294002,salidroside & SB203580,salidroside & U0126,respectively,rMSCs induced for day 7 and day 14,were harvested to analyze the expression of AFP and ALB,which are hepatic proteins.Result:(1)rMSCs of negative control still showed fibroblast-like grew as a whirlpool and gradually evolved into a state of overgrowth,rMSCs induced showed the state of intensive growth and tended to form circular or irregular shapes which were similar to hepatocytes of intensive growth.RT-PCR detection showed that hepatocyte-related genes such as HGF,c-MET,HNF-3β,CYP2B1,AFP,Albumin, CK18 and transferring were expressed in a time-dependent manner and each production of group C was significantly higher than that of group A.Western Blot analysis showed that hepatocyte-specific protein AFP(only expressed in earlier period),ALB,and CK18 were expressed in a time-dependent manner and the level of each protein was significantly higher than that of group A.The levels of aminotransferase,induction of P450 activity,cellular uptake of LDL and urea also increased in a time-dependent manner,which were significantly increased in comparison to group A.(2) Salidroside (10μM and more for salidroside) alone could differentiate towards hepatocytes whereas the differentiation rate of rMSCs presents a platform in the concentration range(20μM more for salidroside).The liver-specific proteins AFP and ALB were expressed in a dose-dependent manner,but 20μM more for salidroside did not possess a stronger induction capacity,but no obvious cell toxicity.(3) rMSCs all grew badly when medium was supplemented with each inhibitor but expression level of AFP and ALB among groups had a statistical significance:rMSCs of group C expressed AFP and ALB protein to some extent;ones of group D expressed more AFP and ALB than that of group C,while ones of group E did not express AFP and ALB protein.Conclusion:salidroside in association with FGF-4 could differentiate rMSCs towards hepatocytes.Differentiated rMSCs have typical functional hepatic characteristics,such as gene expression of HGF,c-MET,HNF-3β,CYP2B1,AFP,Albumin,CK18 and TTR,protein expression of alpha fetoprotein(AFP),albumin(ALB) and CK18,CYP450-dependent activity and inducibility,cellular uptake of LDL and urea synthesis.Salidroside(10μM and more for salidroside) alone could differentiate towards hepatocytes whereas the differentiation rate of rMSCs presents a platform in the concentration range(20μM more for salidroside).Liver-specific proteins,AFP and ALB were expressed in a dose-dependent manner,but 20μM more for salidroside did not possess a stronger induction capacity.Overall,the ERK1/2 and PI3K signaling pathway plays an important role in the regulatory effects of salidroside on hepatic differentiation of rMSCs and involved in cell fate determinations.Chapter four:MSCs could reduce fibrosis or improve function in a rat model of severe chronic liver injury and salidroside has synergistic effects in vivoAim:to investigate rMSCs and rMSCs in associate with salidroside for use in the reversal of experimental liver fibrosis in rat.Methods:Hepatic fibrosis of rats was induced by intraperitoneal administration of porcine serum twice a week for 8 weeks.Control rats received saline instead of porcine serum all the time(8 wks),the others intraperitoneally received porcine serum all the time.rMSCs were administrated by tail vein injection on day 29 while salidroside was intraperitoneally injected once a day from day 29 to day 56.Seven groups were designed:A and B represent group control and model received porcine serum;C,D and E represent group received porcine serum and 5^*10⁶,1^*10⁶ and 5^*10⁵ rMSCs, respectively;F represents group received porcine serum,5^*10⁶ rMSCs and salidroside (40mg/kg);G represents group received porcine serum and salidroside(40mg/kg).All rats were sacrificed on day 56,and then many analyses,such as morphologic and histopathologic analyses,immunofluorescence and fluorescence fluorescence detection of liver sections,plasma levels of total protein(TP),ALB,alkaline phosphatase(ALP), aspartate aminotransferase(AST),alanine aminotransferase(ALT) and transforming growth factor(TGF)-β1,hydroxyproline content assay,were carried out.Results:The amount of collagen deposition in liver of rats of administration of rMSCs(in associate with salidroside or not) was lower than that of rats without administration of MSCs or salidroside,and salidroside has the synergistic effect.Liver sections stained with HE and azan revealed large areas of fibrosis and hepatocyte denaturation in the group B when in comparison to ones of group A.The amelioration was found in the group C,D,E,F and G while liver fibrosis and hepatocyte denaturation in group F were significantly suppressed when in comparison to ones in group C,D,E or G,and salidroside has the synergism effect.Some positive rMSCs were found in group C,D,E and G after transplantation for 28 days,part of which had differentiated towards hepatocytes,but it was difficult to measure the basal rate of cell proliferation and differentiation because rMSCs transplanted were small probability events.Serum ALT and AST activity of group C,D,E,F and G decreased,and ones of group F did to the normal levels when coadministration of salidroside and rMSCs, which suggested salidroside had a synergistic effect.Hepatic hydroxyproline contents in group B were two-fold higher than in normal untreated rats,which indicates the onset of liver fibrosis,rMSC administration could decrease the hydroxyproline content of livers and salidroside had the synergism effect.The levels of serum TGF-β1 increased significantly after intraperitoneal administration of porcine serum for 8 wk.rMSCs transplanted could decrease the level of serum TGF-β1 and,what is best of all, co-administration of rMSCs and salidroside could did it.Conclusion:rMSCs could ameliorate experimental liver fibrosis,and decrease the serum ALT and AST activity and levels of serum TGF-β1 and,what is best of all, rMSCs in association with salidroside could do the same.Suppression of TGF-β1 played an important role in the process of recovery from hepatic fibrosis.