| Background:Acute liver failure has become the hotspot and difficulty of the liver disease research, because of its fast expanding, lots of complications, difficult treatment, high mortality, bad prognosis. Liver transplantation is an effective therapeutic method. However, liver transplantation is limited by a lot of problems, such as insufficient liver donor, severe surgical injury, immunity rejection, high cost and so on. Looking for other effective methods to treat acute liver failure is very important. At present, hepatocyte transplantation, bioartificial liver and hepatic tissue engineering are also limited by the lack of cell sources.The research of stem cells has provided hopes for the treatment of acute liver failure. Stem cells have the ability of renewal and differentiation into at least one mature cell type. In certain conditions, stem cells can differentiate into multiple cells, even the tissues and organs. Mesenchymal stem cells (MSCs) attract more and more attention for their predominance. MSCs have the same basic characteristic with the common stem cells in the ability of renewal and differentiation. MSCs can differentiate into not only mesodermal tissuses but also ectodermal and endodermal tissues. MSCs can be obtained from adult bone marrow and be amplified in vitro. MSCs can not be limited by MHC. For above the merits, MSCs have golden prospect.Research has identified that MSCs have the ability to differentiate into hepatocyte in vitro and in vivo. In 1999, Petersen et al. found that bone marrow cells of rat can differentiate into hepatic stem cells, and differentiate into hepatocyte and biliary epithelial cells, which identified the bone marrow source of hepatic stem cells. Makowka L et al. reported that the transplantation of bone marrow cells in the peritoneal were effective in D-galactosamine induced acute liver failure rats. Allen et al. reported that the transplantation of bone marrow cells could amend the liver function of the mice with Wilson disease. All the studies have identified that bone marrow stem cells can be used to treat many kinds ofliver diseases including acute liver failure. With the development of the research of bone marrow stem cells, It is possible that bone marrow stem cells can be used to treat acute liver failure. Bone marrow stem cells involve hematopoietic cells and mesenchymal stem cells (MSCs). Whether MSCs can be used to treat acute liver failure or not. And what is the mechanism of MSCs treating acute liver failure. We devise the experiment to search the treatment and the probable mechanism.Objective:1. To study the method of separation, cultivation and expandation of MSCs from mice bone marrows. Establishing a stable MSCs'cell lineage of mice for the further study.2. To establish the models of acute liver failure of mice by feeding with CCl4, MSCs cultivated in vitro were transplanted into the acute liver failure mice by tail veins to study the therapeutic effect.3. To discuss the probable mechanism by marking the MSCs in vitro, observing the migrating and homing of MSCs in the liver, detecting the expression levels of TGF-β1 and ALB of the liver tissues.Methods:1. MSCs were isolated, expanded and purified by density gradient centrifugation combined with adherent culture. The growth curves of different passages MSCs were drawn by MTT. The cell cycle was analyzed by flow cytometry. The surface antigen expressions of MSCs were detected by flow cytometry.2. The experimental mice were fed with 40% CCl4 0.05 ml, 0.1 ml, 0.15 ml, 0.2ml, per 10 gram body weight in turn. Observe the statue of their hair, status of spirit, play, appetite, their urine color and the survival rate of the mice. Check the levels of serum ALT, AST and TBIL. The pathological changes of liver tissues were observed by microscope after stained with HE. Establish the stable mice models of acute liver failure treated with CCl4.3. The acute live failure mice were randomly divided into control and treatment groups. MSCs(1×105/0.4ml) were transplanted into tail veins of mice in treament group. Saline was transfused into the tail veins of mice in control group. Determine the levels of serum ALT, AST and TBIL. The pathological changes of liver tissues were observed by microscope after stained with HE. Survival rate was defined as the percentage of surviving animals at any specific time point.4. DAPI-labelled MSCs(1×105/0.4ml) were transplanted into tail veins of acute liver failure mice and normal mice. On day 1,5,10 after transplantation, The distribution of the DAPI-labelled cells were observed by laser scanning confocal microscope.MSCs(1×105/0.4ml) were transplanted into tail veins of mice in treament group. Saline was transfused into the tail veins of mice in control group. Determine the expression levels of TGF-β1 and ALB of the liver tissues on day 1,2,3,4,5,7.5. One-way analysis of variance and T test were adopted to compare everyindex values by SPSS 14.0 statistic analysis software. Survivorship curve was drawed by Kaplan-meier, and the survival rate was analyzed by Log-rank test. Results:1. The MSCs exhibited a small round shape after fresh separation. After cultivated and passaged, the MSCs were homogenenously fusiform shaped. The growth curves of P1, P2 and P3 MSCs were"S"shape. The cells of G0-G1 stage account for 75.27%. The expression of CD 44 was positive, while the expression of CD34 was negative.2. After fed with CCl4, the difference of liver function and survival rate was obvious(P<0.05). There were clinical manifestations and pathological changes of acute liver failure in the group of 0.15ml, per 10 gram body weight. The levels of serum ALT, AST and TBIL in mice fed with CCl4 for 48h were significantly elevated compared with those in control group(11357.70±1180.63 vs 42.88±4.11;12089.36±1847.59 vs 126.11±7.68;122.56±10.76 vs 3.12±0.78, P<0.01). Pathological changes: liver bulk augmented or reduced, liver became yellow, tender. There were multiple necrosis of hepatocyte and infiltration of inflammatory. The configuration of liver lobule vanished, there were no fibre hyperplasia and hepatocyte regeneration.3. The levels of serum ALT, AST and TBIL were decreased after transplantation of MSCs compared with those in control group. 48h:(923.62±98.48 vs 11325.41±1188.26;1180.04±140.67 vs 12057.65±1390.35;23.36±3.23 vs 120.07±12.01,P<0.01). The survival rate of treatment group was 65%, while that of control group was 25%. The survival rate of treatment group was higher than that of control group obviously (P<0.01). The degree of pathological changes of liver in treatment group was lighter than that in control group on day 1 and 2. And the pathological amending of liver in treatment group was more evident than that in control group on day 7.4. After transplantation of DAPI labelled MSCs, DAPI labelled cells were found in the liver of the liver failure group, and were not found in the normal liver on day 1, 5, 10.After fed with CCl4, the expression levels of TGF-β1 in the liver tissues of treatment group started to increase on day 1, and arrived the climax on day 3, started to decrease on day 4. The expression level of TGF-β1 of treatment group was lower than that of control group (P<0.05). The expression level of ALB of treatment group was higher than that of control group on day 7 (P<0.05).Conclusions:1. The method of density gradient centrifugation combined with adherent culture could isolate MSCs from bone marrow simplely. DMEM-LG medium supplemented with 15%fetal bovine serum is suitable for the culture of MSCs.2. The cells of G0-G1 stage account for 75.27% in the 3th passage of MSCs, and the P3 MSCs express CD 44, and did not express hematopoietic cell surface marker CD34. The cultured MSCs lineage is stable and can be used for further research.3. We can establish the stable models of acute liver failure by feeding with 40% CCl4 0.15ml, per 10 gram body weight in mice.4. The intratail vein transplantation of MSCs could amend the injuried liver, lighten the pathological changes, enhance the survival rate in mice. The transplantation of MSCs could treat acute liver failure in mice.5. The probable mechanism of the transplantation of MSCs treating acute liver failure:(1) MSCs could migrate and settle down in the injuried liver, and transformed into the functional hepatocyte.(2) Decrease the expression level of TGF-β1 of injured liver, promote the regeneration of liver. |
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