| BackgroundCurrently, liver transplantation is still the only definitely effective treatment for the end-stage liver diseases caused by various reasons. However, the widely usage of liver transplantation was severely limited by the shortage of liver grafts, the high cost of surgery and postoperative treatment and the long-term complications after surgery. The hepatic stem cell transplantation provides a new approach for the treatment of end-stage liver diseases. The bone marrow mesenchymal stem cells (BMSCs) are one of the candidates which get lots of attention in both clinical and scientific research since they have lots of advantages. BMSCs are easy to be isolated and amplified with strong self-renewing ability, proliferative ability and differential ability, and which can be autologous transplanted. However, there are many controversies in the following aspects. Firstly, how to get enough BMSCs which can meet the need of transplantion in amount, bioactivity and purity, through an easy and convenient method. Secondly, whether the BMSCs transplanted through spleen can migrate and plant into the liver in the condition of portal hypertension which is often accompanied by the end-stage of liver disease. Thirdly, which factors are involved in the migration of BMSCs to liver. Finally, are there any influences to the survival and functional status of hepatocytes under the condition of co-culture with BMSCs.Objectives1. To explore the feasibility of isolation, culture, and purification of BMSCs in vitro, and to evaluate the differentiation of BMSCs into hepatic progenitor cells in vitro.2. To compare the distribution of BMSCs transplanted through spleen between the liver of normal rats and the rats with portal hypertension.3. To investigate the factors, which affect the migration and planting of BMSCs into the liver preliminarily.4. To evaluate the influences to the survival and functional status of hepatocytes under the condition of co-culture with BMSCs.Methods1. BMSCs were isolated from the bone marrow of Sprague-Dawley rats based on the characteristic of attachment and were amplified and purified though continuous passage for five times in vitro. Then, the BMSCs of 5th passage were cultured in induction medium with hepatic growth factor (HGF) and fibrotic growth factor-4 (FGF-4) for two weeks. Morphological change of induced cells was observed by light microscope and transmission electron microscope. The expressions of alpha fetoprotein (AFP), cytokeratin-18 (CK-18) and albumin (ALB) were detected in the induced BMSCs by Immunofluorescence.2. The recipients were randomly divided into two groups:the experimental group and the control group. The bile ducts of rats in experimental group were ligated and cut, while those in control group were leaving. The rats of both groups were rised for two weeks before the BMSCs tranplantation. The induced BMSCs of 5th passage were labeled by CM-Dil and transplanted to the rats though spleen. After one more week feeding, the rats of two groups were killed and the livers were gained to make paraffin sections. The portal pressure and liver function were measured before the rats were killed. Then the paraffin sections were observed and photographed under a fluorescence microscope. The distributions of cells with red fluorescence were evaluated using Image-Pro Plus 6.0 software by comparing the integrated optical density (IOD) between two groups. The relationship between IOD and portal pressure were analyzed.3.The whole-genome expressions were compared between the liver of experimental group and control group by rat’s gene expression profiling microarray. The cell migration and cell adhesion related genes which expressed differentially were screened, and then verified by Real-time PCR and Western blot analysis.4. Human liver lobes were perfused and digested with HBSS (with and without EDTA) and collagenase, and the free hepatocytes were harvested. Hepatocytes were mono-cultured (Group Mono), directly and indirectly co-cultured with BMSCs which were preconditioned by hypoxia and normoxia (Group D-H, D-N, I-H, I-N). The survival status of hepatocytes was analyzed by MTT assay and the functional status was evaluated through ALB level in supernatant by ELISA.Results1. BMSCs can be isolated and purified successfully by the differential adhesion method. The BMSCs were gradually purified along with the continuous subculture, meanwhile the cell morphology transferred from the irregular or polygonal sharp to uniform fusiform. After passage for five times, the positive rates of CD29 and CD 90 measured by flow cytometry reached up to 96%. The immunofluorescence showed that the BMSCs induced by HGF and FGF-4 expressed AFP, CK-18 and ALB, while the cells which were not induced didn’t expressed these markers. The sharp of BMSCs after induction turned to be cubiform and the cell arrangement tended to be tessellated. No significant differences in organelles between the 1st and 5th passage BMSCs were noted under a transmission electron microscope. However, the Golgi apparatus, ribosomes, and endoplasmic reticulum increased significantly in the induced BMSCs.2. The induced BMSCs can be labeled with red fluorescence in the cell membrane after stained by CM-Dil, with no influence to the attachment, growth and proliferation abilities. Two weeks after common bile ducts ligation, a series of clinical manifestations of biliary obstruction, including auricle and tail jaundice, deep yellow urine, off-white stool, and emaciation, were developed. Three weeks after common bile ducts ligation, liver function tests showed that the total bilirubin (TB), the direct bilirubin (DB), the alanine aminotransaminase (ALT) and the aspartate aminotransferase (AST) were much higher in experimental group than in control group (P<0.01), while the albumin (ALB) was lower in experimental group than in control group (P<0.05). All survival rats in the experimental group were found cysts at the porta hepatis, and all livers were clearly enlarged and had sclerosis with many small nodules. The portal pressure of the experimental group was significantly higher than that of the control group (18.04±2.35 vs.9.75±1.40cmH2O, P<0.01). The IOD of the experimental group was much larger than that of the control group (1130067±209464 vs.293505±88383/Hpf, P<0.01), which showed that more transplanted cells migrated and planted into the liver of the experimental group.3. The expression of the four genes CX3CL1, CXCR4, MLLT4 and PDGFA which associated to cell migration and adhesion, were up-regulated in experimental group than in control group showed by rat’s gene expression profiling microarray. The results of Real-time PCR showed that the mRNA level of CX3CL1 and CXCR4 were much higher in experimental group than in control group (P<0.01), while the mRNA level of the other two genes had no statistical difference between the two groups (P>0.05). This result was further verified by western blot assay at protein level:the proteins of CX3CL1 and CXCR4 were higher in experimental group than in control group (P<0.05).4. The hepatocytes survival in group I-N and D-N was higher than in group Mono at the 4th and 7th co-culture day (P<0.05). The hepatocytes survival were higher in group I-H than in group I-N, and in group D-H than in group D-N respectively at the 4th co-culture day(P<0.05). No difference was detected between group I-N and group Mono in ALB level through the whole co-culture process (P>0.05), while ALB level in group D-N was higher than in group Mono at the 4th co-culture day (P<0.05). The ALB level in group D-N was higher than in group I-N at the 4th co-culture day (P<0.05), and that in group D-H was higher than in group I-H at the 4th and 7th co-culture day (P<0.05). There was no difference between group I-N and I-H, group D-N and D-H in ALB level through the whole co-culture process (P>0.05).Conclusions1. The combination of HGF and FGF-4 induces the differentiation of BMSCs into hepatic progenitor cells, which express the markers of hepatic cells AFP, CK-18, and ALB. The differentiated cells are different from BMSCs morphologically in micro structure and ultrastructure.2. CM-Dil is an ideal tracer for cell transplantation. Two weeks after the common bile ducts ligation, the rats presented various symptoms of jaundice, and one more week later, the models of biliary cirrhosis and portal hypertension were developed. The recruitment of BMSCs (after transplantation in the spleen) was improved in rats with portal hypertension. This result indicates that, there must be some chemical factors driving the transplanted cells migrate to the injured liver.3. The expressions of CX3CL1 and CXCR4 in liver of experimental group are higher than in control group at both RNA and protein level. CX3CL1 and CXCR4 might be associated with the migration of BMSCs to injured liver.4. The hepatocytes survival can be elevated by direct and indirect co-culture with BMSCs, and no different effects are observed between these two methods. The survival of hepatocytes could benefit more from the hypoxia preconditioned BMSCs than the normoxia preconditioned BMSCs. No influence to the ALB secretion was observed under the condition of indirect co-culture with BMSCs, while the ALB secretion was up-regulated when the hepatocytes were directly co-cultured with BMSCs. Whether the BMSCs were preconditioned by hypoxia gains no difference to the influence to ALB secretion. |
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