Research On Bone Marrow Stem Cells Transplantation For The Treatment Of Acute And Chronic Liver Injury

Background and aim:At present,the treatment for acute liver failure and terminal stage liver disease are tough problems of worldwide. OLT (orthotopic liver transplantation) is the most effective method ,but there are still some difficulties with widely clinical application due to insufficient liver donor, high cost ,immunity rejection and other reasons. Hepatocellular transplantation, bioartificial liver and hepatic tissue engineering,which have shown the potential for the treatment of liver diseases, are limited by the lack of liver cells. The status encourage the search for new therapies.The development of stem cells provides new insights into therapies of liver disease.Mesenchymal stem cells (MSCs) from bone marrow are capable of differentiation into mature cells of multiple mesenchymal tissues including fat, bone, and cartilage. Diverse studies have suggested that MSCs might have the ability to differentiate into cell types other than those of the tissues in which they reside or derive during embryonic development. For instance, MSCs are known to differentiate into neural cells and hepatic cells under appropriate experimental conditions. From 1999, the Journal of Science and Nature reported that bone stem cells have been shown to differentiate into liver cells lineage in rodents and patients who received orthotopic liver transplantation or bone marrow transplantation. The potential of MSC differentiate into hepatocytic cells provides alternative cells source for hepatocellular transplantation, bioartificial liver and hepatic tissue engineering, and open new possibilities for therapies of liver disease.Although the studies about MSCs on liver disease have obtained an enormous development, there are several questions remain with regard to their potential therapeutic use. The first problem is how to separate, culture and proliferate MSCs in vitro? Secondly, how to establish the appropriate experimental conditions inducing differentiation of MSCs into hepatocyte in vitro. Then, how to determine the number of cells and route of transplantion, and so on. This study aim to establish a method of isolation and expandation mesenchymal stem cells, to optimize and standardize the condition of inducing MSCs to differentiate into hepatocyte-like cells in vitro, and to investigate the induced MSCs transplantation for the treatment of acute and chronic liver injury treated with CCl4. The exploration will be beneficical for clinical application.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 surface antigen expressions of MSCs were detected by flow cytometry. The ultramicrostructure of MSCs were observed by transmission electron microscope.2. MSCs were induced to differentiate into hepatocyte-like cells with DMEM–LG medium, containing 10% FBS, ITS, 20ng/mlHGF, 10ng/ml FGF-4, 10ng/ml EGF. on 7d, 14d and 21d after induction, The mRNA expression of AFP and ALB were analyzed using RT-PCR. The markers of hepatocyte lineage were examined by immunocyte chemistry. The changes of ultramicrostructure of MSCs were observed by transmission electron microscope.3. The BALB/c mice were treated with 0.2% CCl4 peanut oil mixture by intraperitoneal injection (20ml/kg) to establish acute liver injury model. The mice were randomly divided into control and treatment groups. DAPI-labelled MSCs on 21th day after induction (1×105/0.3ml) were transplanted into the spleen of mice in treament group.PBS was transfused into the spleen of mice in control group. On day 3 and 7 after transplantation, The distribution of DAPI-labelled cells were observed by fluorescence microscopy. The liver function indexes and pathologic histology of liver were detected at same time.4. The Wistar rats were treated with 40% CCl4 peanut oil mixture by hypodermical injection (0.5ml/100g at the first time, then 0.3ml/100g every 3 days for 8 weeks) to establish chronic liver injury model.The model rats were randomly divided into control 1, 2, 3 groups and treatment 1, 2, 3, groups. DAPI-labelled MSCs on 14th day after induction (1×106/0.5ml) were transplanted into the rats by portal vein, spleen and tail vein, respectively. PBS was transfused into rats in various control groups. On day 7 and 14 after transplantation,The distribution of the DAPI-labelled cells were observed by fluorescence microscopy.The liver function indexes and pathologic histology of liver were detected at same time.5. One-way analysis of variance and t test were adopted to compare every index values by SPSS 12.0 statistic analysis software.Results:1. The MNCs exhibited a small round shape after fresh separation.The rate of living cells was above 95%. The MSCs were homogenenously long fusiform shaped, fibroblast-like 10-13 days after cultivation. The cell population increased 1.8-2.4 times by amplification per passage.The grow curves of P1, P3 and P5 MSCs were"S"shape. The positive expression ratio of CD44, CD 29 were 80.22%, 87.83%, respectively,while near negative for hematopoietic cell surface marker CD34, CD45. The ultrastructure of MSCs indicated that there are two shape. One at quiescent state, with high nuclear-cytoplasmic ratio and few cytoplasmic organelles;other at active state, with microvilli on the surface,low nuclear-cytoplasmic ratio and many cytoplasmic organelles.2. The morphology of MSCs transformed gradually into round,polygonal endothelial cell shape after induction, resembling as hepatocytes. AFP mRNA and protain were detected on 7th and14th day after induction. The expression of AFP mRNA on 7th day was higher than that on 14th day (P<0.05). ALB mRNA and protain were detected on 14th and 21th day after induction.The expression value of ALB mRNA on 21th day was higher than that on 14th day.CK18, Hepatocyte antigen were detected on 14th and 21th day after induction. The markers of hepatocyte lineage did not appear in no growth factor induction group. The positive expression ratio of ALB, CK18 and hepatocyte antigen were 88.6%, 81.2% and79.6%, respectively. The ultrastructure of MSCs indicated that cells grew bigger in size with similar round nucelus, large nucleoli,and a lot of endoplasmic reticulum, mitochondrion, lysosome,glycogen granule.DAPI labelled nuclei exhibited the bright blue fluorescence.. DAPI labelled efficiency achieved close to 100%.There are no obvious side effects for DAPI labelled cells.3. The levels of serum ALT and AST in mice treated with with CCl4 for 24h were significantly elevated compared with those in normal (191.80±6.96 vs 50.37±11.60; 289.30±8.97vs 105.92±16.83, P<0.01). Compared with control group, the levels of serum ALT and AST were decreased on day 3 and day 7 after transplantation (121.43±6.05vs 157.22±8.89; 178.33±16.01vs 238.33±9.98, P< 0.01). DAPI labelled cells were found in the spleen and liver in group T. DAPI labelled cells were not found in group C.Pathological changes of liver displayed multiple necrosis of hepatocyte, diffuse degeneration of hepatocyte and infiltration of inflammatory cells in mice treated with with CCl4 for 24h. The severity of pathological changes of liver in group C were more extensive than those in groupT on day 3 and day 7 after transplantation.4. The levels of serum liver function index in rats treated with CCl4 for 8 weeks were significantly elevated compared with those in normal.Compared with control group, the levels of serum ALT, AST, GGT and TBil were decreased on day 7and day 14 after transplantation. DAPI labelled cells were found in the spleen and liver in groupT1, T2and T3. DAPI labelled cells were not found in control group.Pathological changes of liver displayed significant necrosis, steatosis, inflammatory cell infiltration and fibrosis in rats treated with with CCl4 for 8 weeks. Compared with control group, The severity of Pathological changes of liver were alleviated in various treament group on day 7 and day 14 after transplantation. The pathologial scores of chronic liver injury on 14thd after transplantation of induced MSCs had statistically difference among group T1, T2 and T3 (P<0.05). The epithelioid cells with large nucelus and plentiful endochylema in spleen in group T2 were detected the positive expression of ALB and hepatocyte antigen.Conclusions:1. It is a simple and practical method to separate MSCs from the bone marrow by means of density gradient centrifugation and adherent culture. DMEM-LG medium supplemented with 10% fetal bovine serum is suitable for culture of MSCs.2. The 3th passage of bone marrow MSCs strongly express CD44 and CD 29, while near negative for hematopoietic cell surface marker of CD34, CD45. The 3th passage of bone marrow MSCs is suitable for inducing to diffentiate into various cell lineage.3. Both rats and mice MSCs could be differentiated into a liver cell lineage. DMEM-LG medium supplemented with 10% FBS, ITS, 20ng/ml HGF, 10ng/ml FGF-4 and 10ng/ml EGF is sufficent to induce bone marrow MSCs differentiate into a liver cell lineage.4. DAPI is convenient for labelling MSCs in vitro. DAPI labelled cell could be used to trace in vivo for about 2 weeks.5. Acute liver injury model in mice could be established by intraperitoneal injection with 0.2% CCl4 peanut oil mixture (20ml/kg). Induced bone marrow MSCs could survive,settle down in mice spleen, and migrate into liver after transfusion.Intrasplenical transplantation of induced bone marrow MSCs ameliorate liver function of acute hepatic injury.6. Chronic liver injury model in rats could be established by hypodermical injection with 40% CCl4 peanut oil mixture (0.5 ml/100g at the first time,then 0.3ml/100g every3 days for 8 weeks). Induced bone marrow MSCs could migrate into liver after intraportal vein, intrasplenical and intratail vein transplantation, and ameliorate liver function of chronic hepatic injury. The route of intrasplenical transplantation is better than intraportal vein and intratail vein transplantation .