The Effects Of TGF-β1 On The Mesenchymal Stem Cells Transplantation On Hepatic Fibrogenesis And Hepatoma In Rats

ObjectiveOn the base of establishment of bone mesenchymal stem cell's culture and amplification system in vitro,to investigate the effects of mesenchymal stem cells (MSCs) transplantation at different times,different durations as well as different transplantation ways on hepatic fibrogenesis in rats.And the possible mechanisms of bone mesenchymal stem cells transplantation on hepatic fibrogenesis and hepatoma in rats were pursuit from the point of TGF-β1.Methods1.Combined with stationary culture MSCs of male SD rat were isolated and purified by density gradient centrifugation and identified by inductions.2.The forth passage cells(5×10⁶/peace),were respectively injected into the homogeneous female rats via the caudalis vena on the sixth week and the eighth week of the hepatic fibrosis model induced by carbon tetrachloride(CCL4) in rats.Four and six weeks after MSC transplantation,the living state of rats and pathology of the liver in transplanted and control groups were evaluated by Scheuer inflammation activity score and Schmid M fibrosis grading score respectively.3.The forth passage cells(5×10⁶/peace),were respectively injected into the homogeneous female rats via intraperitoneal injection, the caudalis vena and intrahepatic injection on the sixth week of the hepatic fibrosis model induced by carbon tetrachloride(CCL4) in rats.Six weeks after MSC transplantation,the living state of rats and pathology of the liver in transplanted and control groups were evaluated by Scheuer inflammation activity score and Schmid M fibrosis grading score respectively.Also,the activation stage of HSC was detected by immunohistochemical.4.With the establishment of the hepatic fibrosis model and hepatoma model induced by carbon tetrachloride(CCL4) and diethylnitrosamine respectively in rats,the effects of TGF-β1 on the mesenchymal stem cells transplantation on hepatic fibrogenesis and hepatoma in rats were investigated.Results1.Combined with density gradient centrifugation homogeneous MSCs of male SD rat were isolated,purified and passaged by stationary culture.Also,they were identified by induced into lipocyte and osteoplast.2.On 6^th week of MSCs transplantation,no difference in inflammation activity grade was found in both ten weeks model groups.The mean value of Scheuer inflammation score was 3.58 and 3.73,respectively(u=-0.421,P=0.853).While the mean value of Schmid M fibrosis score was 4.75 in the transplantation group and 5.54 in control group u=-2.173,P=0.041).While in twelve model groups with the same MSCs transplantation on 6^th week,there were obviously differences in pathology of the liver. The mean value of Scheuer inflammation score was 0.42 and 3.17,respectively (u=-4.218,P < 0.01).And the mean value of Schmid M fibrosis score was 1.58 in the transplantation group and 5.12 in control group(u=-3.653,P<0.01).Differences were also found in 6^th week of MSCs transplantation groups with different transplantation times,4 weeks and 6 weeks respectively.The mean value of Scheuer inflammation score was 3.58 and 0.42,respectively(u=-4.214,P < 0.001).While the mean value of Schmid M fibrosis score was 4.75 in the transplantation group and 1.58 in control group(u=-3.79,P<0.011).In twelve weeks model groups with MSCs transplantation on 8^th week,there were no significant differences in inflammation activity grade and fibrosis stage between transplantation group and control group(u=-0.421,P=0.860; u=-0.352,P=0.732).But with the different transplant time,6^th week and 8^th week, significant differences were found in both twelve weeks model groups.The mean value of Scheuer inflammation score was 0.42 and 3.64,respectively(u=-4.146,P < 0.01).While the mean value of Schmid M fibrosis score was 1.58 in the 6^th week transplantation group and 5.27 in the 8^th week transplantation group(u=-3.88, P<0.01).3.Six weeks of MSC transplantation later,all rats were killed.No differences of survival rate were found in all groups,but there were obviously differences of ascites incidence in all groups,50%in control group,40%in intraperitoneal injection group, 0%in caudalis vena injection group and 18.18%in intrahepatic injection group. Between control group and intraperitoneal injection group,no differences were found in inflammation activity grade,fibrosis grade and the activation stage of HSC(u=-1.58, P=0.11,u=-0.82,P=0.41,u=-1.37,P=0.17).While the mean value of Scheuer inflammation score,Schmid M fibrosis score and the activation score of HSC between control group and caudalis vena injection group were obviously different, with 3.58 and 0.42(u=-4.22,P < 0.01),5.17 and 1.58(u=-3.65,P < 0.01),2.25 and 0.58(u=-2.93,P < 0.01),respectively.The same differences were also found between control group and intrahepatic injection group,with the mean value of 3.58 and 1.27(u=-3.13,P < 0.01),5.17 and 3.00(u=-2.54,P < 0.01),2.25 and 0.55(u=-3.11,P < 0.01),respectively.All the data were further analyzed between different transplantation groups.Obviously differences were found in inflammation activity grade,fibrosis grade and the activation stage of HSC between intraperitoneal injection group and caudalis vena injection groups(P < 0.05),while no difference between caudalis vena injection group and intrahepatic injection group.The rate of shaped cells were also different in all groups,50%in control group,30%in intraperitoneal injection group,0%in caudalis vena injection group and 9.1%in intrahepatic injection group.4.Based on the hepatic fibrosis model induced by carbon tetrachloride(CCL4),the mean value of Scheuer inflammation score,Schmid M fibrosis score and the activation score of HSC between control group and intervention group were obviously different,with 3.58 and 0.42(u=-4.22,P < 0.01),5.17 and 1.58(u=-3.65,P < 0.01), 2.25 and 0.58(u=-2.93,P < 0.01),respectively.The contents of TGF-β1 were also different between two groups(F=11.024,p=0.029).However,no differences were found in the contents of smad4 and smad7 between two groups(F=0.178,p=0.695, F=0.00,p=0.986).On the hepatoma model induced by diethylnitrosamine,the cell cycle was restrained in intervention group and most cells were in G₁ phase.While in control group,most cells were in S phase(G₁ phase P =0.000,G₂ phase P=0.000,S phase P=0.000).However,there was no difference in the incidence of liver cancer between two groups.Further more,the mean value of Scheuer inflammation score, Schmid M fibrosis score and the activation score of HSC between two groups had no differences.The same results were also found in the contents of TGF-β1,smad4 and smad7(F=1.538,p=0.283,F=4.989,p=0.298,F=2.698,p=0.176).Conclusion1.The method that combined with stationary culture and density gradient centrifugation was an effective way to isolate and purify BSMC.2.Early MSCs transplantation can alleviate inflammation reaction and improve fibrosis stage in transplanted rats induced by CCL₄.And the improve degree of inflammation reaction and fibrosis stage has a positive correlation of different times as well as the duration on hepatic fibrogenesis.3.MSC transplantation via the caudalis vena and intrahepatic injection has the same effect on alleviating inflammation reaction and improve fibrosis stage in transplanted rats induced by CCL4 MSC transplantation could lessen the activation stage of HSC and the incidence of special-shaped cells in cirrhosis.4.The study suggests that BMSC transplantations probably by inhibiting the secretion of TGF-β1,reduce hepatic stellate cells activation and thus slowing the process of liver fibrosis.In carbon tetrachloride-induced rat liver fibrosis model,BMSCs possible regulate cell cycle by reducing the content of TGF-β1,to reduce the incidence of special-shaped cells,but in diethylnitrosamine-induced rat liver cancer model,TGF-β1 is not the way that BMSCs regulate cell cycle.