The Effects And Mechanism Of Mesenchymal Stem Cells In Liver Fibrogenesis

【Background and Objective】Hepatic fibrosis, characterized by deposition of extracellular matrix (ECM) in the Disse's space, is a pathological response to a variety of chronic liver diseases, which is recognized as a dynamic and reversal process. Therefore, blocking, inhibition or even reversal of hepatic fibrosis is a major target for the treatment of chronic liver disease. Generally, it has been accepted that activated hepatic stellate cells (HSC), accompanying phenotypic transformation into myofibroblast-like cells, play the pivotal role in hepatic fibrogenesis. In the past decades, it is an important target on hepatic fibrosis therapy by repressing activation, proliferation and migration of HSC as well as inducing its apoptosis. Recently, several studies reported that in vivo environment by simply inhibiting the effect of the treatment of liver fibrosis is not ideal. And more researchers have come to realize that HSC is not the only important influence on the cells of liver fibrosis. So researchers take more attention to find other cell subsets that involve in hepatic fibrogenesis, and study the mechanism of these cells.The development of Liver fibrosis is always accompanied with chronic inflammation. The cells in liver will secrete a large number of cytokines, growth factors, chemokines involved in fibrosis during liver damage. At the same time a variety of extrahepatic cells will also chemotactic to the injured parts to involve in fibrogenesis. Has been shown that mesenchymal stem cells (MSCs) are one of these extrahepatic cell populations, during liver injury they also chemotaxis to the site of injury involved in hepatic fibrogenesis. MSCs is a pluripotent differential non-hematopoietic stem cells, generally located in the bone marrow. The understanding to MSCs in the past mainly focused on the capability of chemotaxis to the injuried site and the function of repairing damage, so as to develop its as a gene drug. In some diseases, such as connective tissue diseases and bone injury, MSCs therapy achieved good results. But with further research, the investigators found that MSCs in some diseases, also may play some adverse effects, such as it can promote the growth and development of various tumors. For many diseases, MSCs may be play a role of double-edged sword. These results suggested that the researchers should be re-examine strictly the role of MSCs in the disease.Currently, there is a big controvery to the role of MSCs in hepatic fibrogenesis. The purposes of our study is to explore the effect of MSCs to fibrogenesis is whether promoting or inhibiting.Hepatic fibrosis and other parts of the fibrosis is that the biggest difference between the vascular system reconstruction. Reconstruction of the vascular system associated with the development of liver fibrosis has always been. In this process must secrete large amounts of liver and angiogenesis-related cytokines, such as VEGF, IL-6, IL-8 and so on. And some researchers also confirmed in vitro VEGF, PDGF and other angiogenic some cytokines or growth factors can promote the proliferation of MSCs. MSCs to the damaged parts of the chemokine is a continuous process, as long as the cause of the existence of fully established inflammatory environment, MSCs will be a steady stream of chemotaxis to the liver, bone marrow, MSCs therefore have to supplement self-proliferation. On this basis, we hypothesize, the secretion of hepatic fibrosis in a large number of angiogenesis-promoting cytokines such as VEGF in vivo bone marrow MSCs may be a major reason for continued proliferation.Of course, MSCs migrate from extrahepatic tissues to the liver, in addition to the proliferation of MSCs been added, but also the ability of MSCs with directional migration. Studies have shown that MSCs migration and chemokine receptor family members and their closely related, the most important is CC-chemokine family and CXC-chemokine family. But there are also studies have shown that different organs or tissue damage, the impact of the migration of MSCs chemokine and its receptor is not the same, so clear which chemokines in liver injury, liver fibrosis in the liver of MSCs the chemotactic recruitment, we can control the development of fibrosis to find new therapeutic targets. The issue in the establishment of a variety of animal models based on the mouse, using fluorescent staining, immunohistochemistry, PCR and other molecular biology techniques to explore the formation of MSCs in the liver (at least the early fiber formation) played the and its effect on liver function. While in-depth discussion of the BM-MSCs in vivo proliferation, migration, raising the main reason for its mechanism. Combined with the detection of liver cirrhosis clinical samples to further understand the pathogenesis of hepatic fibrosis, hepatic fibrosis is to provide a new way of thinking.In this study ,we used a variety of animal models in mice, based on a clear process of liver fibrosis in MSCs in the presence of liver tissue and the formation of fiber (at least the early fiber formation) and liver function. And through a number of experiments in vivo testing, described BM-MSCs in the process of liver fibrosis in the liver migrate to the transfer of the bone marrow, raising the main factors, while exploring the causes of liver damage after the proliferation of BM-MSCs in vivo the main factors and mechanisms to the pathogenesis of hepatic fibrosis research and treatment of new ideas.【Methods】1. Isolation, culture and identification of mesenchymal stem cells1.1 Isolation and culture of MSCsRespectively male WT-BALB / c mice and EGFP-BALB / c mice were isolated and cultured WT-MSCs and EGFP-MSCs cells. By density gradient centrifugation and adherent culture methods to separate the combination of MSCs, but this time also mixed with a small amount of mononuclear cells or the situation, and fibroblasts. Use of these cells and MSCs cultured on plastic bottles of different strength of adherent cells when subcultured with 0.05% trypsin +0.02% EDTA digestion and 2-3min rest after repeated digestion can be gradually removed after the passage of other types of cells.1.2 MSCs IdentificationDynamic inverted microscope WT-MSCs in mice the growth process of primary culture; fluorescence microscope mice EGFP-MSCs cell growth process. Flow cytometry, and immunofluorescence staining to detect surface markers on mouse MSCs CD34, CD90, CD45, CD90, CD105 expression. Osteogenic differentiation Von Kossa staining and ALP activity quantification, detection of directional differentiation of MSCs into the bone ability to adipogenic differentiation Oil Red O staining directional differentiation of MSCs into fat capacity.2. Effects of MSCs on liver fibrogenesisConstruction of mouse CCL4 liver injury model (model 1), and bone marrow transplantation model + CCL4 liver injury model (model 2). Model determined after successful bone marrow transplant bone marrow receptor wt donor mice within the EGFP-MSCs exist. By tail vein injection of exogenous EGFP-MSCs in mice to model 1, 3d, the frozen section was observed under immunofluorescence microscopy exogenous chemotactic parts of MSCs, as well as the degree of liver fibrosis; trace through the model 2 Endogenous EGFP-MSCs in the liver after injury chemotactic parts; also detected at different time points AST, ALT levels, detection of liver fibrosis in the liver function of the situation3. Mobilization of MSCs during hepatic fibrogenesisModel 1 and Model 2 using mice, some mice were sacrificed every week for 5 weeks. Model mice were sacrificed 1 week, 3 days before injection in the death of the EGFP-MSCs exogenous tracer. Frozen sections were observed under fluorescence microscope to determine the intrinsic and extrinsic of MSCs in the mouse liver in animal models of chemotaxis to raise the time. Realtime-PCR Screening MSCs important chemokine surface receptors, and the corresponding damage the liver play a role in the major chemokines. Then observe this chemokine in the mouse liver and the expression of bone marrow and in vivo experiments on BM-MSCs of chemotaxis.4. The proliferative effect and mechanism of VEGF on MSCs during liver fibrogenesisBy immunohistochemistry, realtime-PCR, ELISA used to detect liver and serum VEGF mRNA and / or protein expression. Vitro cell proliferation assay and monoclonal VEGF formation test the impact of the proliferation of MSCs, flow cytometry, mice, VEGF was detected on the proliferation of BM-MSCs, and the use of Avastin can reverse this effect. Transwell experiments and also the use of scratch test whether VEGF can promote the ability of MSCs chemotactic migration.5. Statistical AnalysisStatistical analysis of values was performed with SPSS (11.0 version) and Prism GraphPad5 software, with a P value <0.05 considered significant and P <0.01 as very significant.【Results】1. Isolation, culture and identification of mesenchymal stem cells(1) MSCs isolation and culture of: mouse BM-MSCs were cultured primary cells, 24h after more adherent cells ,48-72h after the spindle into spindle-shaped or growth, the first form of a typical 4-5 day proliferation of uniform distribution of clusters of lesions, 7-10 days non-adherent cells disappeared after repeated medium change, cell fusion more than 80%. After several passages and then cultured spindle cells were tested. MSCs cultured human cell lines cells were observed from the morphology typical spindle cell morphology consistent, tightly arranged similar to the whirlpool.(2) Murine BM-MSCs Identification of surface antigen: memory cells by flow cytometry using immunofluorescence staining to detect the isolated and cultured MSCs, the expression of surface markers showed that the surface expression of adhesion molecules CD29, CD105 , CD90 positive rate of 95% or more, almost no expression of blood cell surface markers CD34, CD45. That these cells were isolated and cultured MSCs characteristics with a very high proportion.(3) A number of differentiation potential of MSCs in mice: induction of differentiation into bone 14d, our ALP-positive cells were detected, compared with the control group found that the induction group quantitative ALP activity increased. Section 21d, Von Kossa staining, cells showed dense black nodules. Adipogenic differentiation 8d, light microscope, morphological changes, intracellular lipid vacuoles lack circular translucent, applied in situ Oil Red-O lipid staining results seen after staining of lipid droplets stained red2. Effects of MSCs on liver fibrogenesis(1) MSCs participate in the formation of hepatic fibrosis: wt mice were injected with exogenous EGFP-MSCs with the blood circulation to the damaged liver, frozen section immunofluorescence figure shows a large number of green fluorescent protein distribution in the fiber spacing nearby. Endogenous EGFP-MSCs in the liver after injury by chemotaxis to the site of injury in liver fibrosis. Human MSCs to SSEA4 markers to detect liver fibrosis in human MSCs tissue showed the expression of the main parts of the fiber spacing and the junction of the liver parenchyma, as markers by flow cytometry, cell separation , most of which are still successful bone cells, the ability to differentiate into fat, the MSCs cells. This indicates that MSCs cells are also involved in the formation of liver fibrosis.(2) Murine bone marrow mesenchymal stem cells promote fibrosis: Sirius red staining, and Masson staining, and quantitative analysis of hydroxyproline found in mice injected with a large number of MSCs was more severe fibrosis in the three control groups (P <0.05). However, liver function (mainly AST) there is some improvement, indicating that MSC to accelerate liver fibrosis is a repair response to injury, to a certain extent, can improve liver function.3. Mobilization of MSCs during hepatic fibrogenesis(1) MSCs to the liver, liver damage during the time of mobilization to raise Want to know BM-MSCs to specific mechanisms of liver chemokine, the first time that MSCs chemotaxis, frozen sections from both the results of animal models to see, MSCs mobilization time in 3 weeks. With this mobilization level over time is also increasing.(2) The process of liver fibrosis screening play a major role of chemokines and their receptors.realtime-PCR assay to the mouse MSCs cells mainly expressed CCR1, CCR2, CCR5, CCR7, CXCR4, CXCR6 and other chemokine receptor 6, and the corresponding receptors of these chemokines, we detected the secretion of liver damage after major liver There CCR1 chemokine ligand MIP1α, MIP1β; CCR5 ligands RANTES, but increased the most obvious is the CXCR4 ligand SDF-1α, we consider the process of fibril formation in the liver to promote the most important chemokine MSCs is SDF-1α/CXCR4 chemotactic axis.(3) Verify SDF-1α/CXCR4 chemotactic chemotactic axis raised on the role of MSCsThe results showed that liver damage the liver of mice during the expression of SDF-1αincreased gradually, while the expression of bone marrow SDF-1αdecreased slightly, when the liver was higher than in the bone marrow SDF-1αconcentration gradient formed only when a large number of mobilized from the bone marrow of the MSCs cells, which explains why the first 3 weeks, to see the large number of green fluorescent protein found in the liver. The Transwell The results also show that SDF-1αin vitro on MSCs do have a strong chemotactic effect.4. The proliferative effect and mechanism of VEGF on MSCs during liver fibrogenesisThe results showed that the process of liver damage, liver and a large number of VEGF production and secretion, which can be distributed with the blood throughout the body. Exogenous VEGF can increase the recipient mice injected with bone marrow content of EGFP-MSCs, and this effect can be reversed VEGF monoclonal antibody Avastin. Transwell test and scratch test results showed that VEGF chemotactic migration of MSC's capability is not strong (P> 0.05). This shows that the mobilization of VEGF in the MSC is to promote a major role in the proliferation of MSCs, rather than the migration of MSCs.【Conclusion】All of our results revealed that:1. The process of liver damage, mesenchymal stem cells promote the formation of fibrosis, at least for early liver fibrosis. 2. The early hepatic fibrosis is mainly due to the repair of liver injury, so if the acceleration of liver repair man for the formation of early stage of liver fibrosis, liver function can be improved correspondingly.3. The process of liver damage, bone marrow mesenchymal stem cells mobilization, mainly depends on the SDF1α/CXCR4 chemotactic axis, the concentration of liver and bone marrow SDF1αMSCs determines the direction of change. This chemotactic axis is moved out of bone with MSCs into the circulation, to the damaged parts of the chemokines, raising one of the main factors.4. MSCs liver fibrosis in chemotaxis to the site of injury is an ongoing process, but also in human liver tissue is still able to detect the presence of MSCs. VEGF is the formation of bone marrow fibrosis and proliferation of MSCs one of the main factors.