The Tumorigenic And Its Gene Expression Changes Of Differential Hepatocytes From Rat Bone Marrow-derived Mesenchymal Stem Cells

Bone marrow-derived mesenchymai stem cells(marrow mesenchymal stem cells, MSCs) is a class of stem cell subsets in bone marrow with multiple differentiation potential.Under certain conditions,it can differentiate into many different tissues or cells.Recent studies have shown that,MSCs were induced by stem cell factor(SCF), hepatocyte growth factor(HGF),fibroblast growth factor-4(FGF-4) and hepatic extract fluid to differentiate into hepatocytes.This study was undertaken to investigate the best conditions to differentiate into hepatocytes,the function characteristics and the safety of cell differentiation during MSCs differentiation into liver cells.At the same time,we applied gene chip to analyze gene expression profiles in the process of cell differentiation and reveal the molecular mechanism of differentiation.This provides experimental evidence for clinical application of MSCs to treat liver disease.【Purpose】1.Compared the inducing effects of current common factors to explore the optimal conditions to induce rat bone marrow-derived MSCs differentiate into hepatocytes.2.The use of pathology and molecular biology technique to detect features of differential cells,comprehensively identified whether differential cells was the hepatocytes.3.Hepatocytes after differentiation and MSCs were injected into the skin of nude mice to observe whether resulted in tumor,and provide secure theoretical basis for the clinical application of MSCs as seed cells in the liver regenerative medicine.4.Application of gene chip to detect the gene expression profiles between normal hepatocytes and MSCs or between normal hepatocytes and hepatocytes after differentiation,in order to understand the molecular mechanism of differentiation.【Methods】 1.We collected the limb bone marrow of adult rats in sterile conditions and cultured by direct screening method or adherent density gradient centrifugation.And the original and third generation and sixth generation MSCs were detect the cell surface markers CD29,CD34,CD90 expression by flow cytometry.2.Different amounts of SCF,HGF,FGF-4,different combinations of cytokines and different amounts of hepatic extract fluid were added to the culture medium to induce MSCs differentiate into hepatocytes,and taking the culture medium without factors as control group.The morphological changes of the induced cells were observed.3.The amount of albumin and urea production in the induced culture supernatant was detected by biochemistry kit.Expression of cytokeratin-18(CK18) in differential cells was detected by immunocytochemistry.And we observed the intake of indocyanine green(ICG) in differential cells.4.Hepatocytes after differentiation induced by single cell factor SCF,HGF and FGF-4,three kinds of cytokines and hepatic extract fluid were respectively injected into the skin of nude mice.After 2 months,the skin tissue of local injection,liver tissue and lung tissues were observed.5.We used gene chip technique to detect the gene expression profiles between normal hepatocytes and MSCs or between normal hepatocytes and hepatocytes after differentiation.The related genes in the process of bone marrow Macs differentiation into hepatocyte were screened.We revealed the molecular mechanism of differentiation from the molecular level.【Results】1.MSCs from isolated culture grew well.Cell morphology was long spindle and arranged in spiral-shaped.MSCs by adherent cultured directly grow faster compared with MSCs by density gradient centrifugation cultured,but MSCs by the density gradient centrifugation cultured was relatively more pure.By flow cytometry showed the original and third generation and sixth generation MSCs were expressed CD29, CD90,and not to express CD34.2.Cells after induced differentiation were round or polygonal cells.These cells were similar to morphology of hepatocytes.The cells without any inducing factors were still long spindle.Different amounts of a single SCF,HGF,FGF-4,different combinations of cytokines and different amounts of hepatic extract fluid can induce MSCs to differentiate into hepatocytes,but hepatic extract fluid(1.0mg/ml) group had the best inducted effect.3.The induced culture supernatants were different amounts of albumin and urea production,differential cell were seen the different degrees expression of CK18 and intake of ICG.cell culture supernatants without any inducing factors were no albumin and urea production.And cells did not express CK18,while not intake of ICG.4.In the 2 months tumorigenicity in nude mice experiments,skin tissue of local injection,liver tissue and lung tissues of nude mice had not format bump by eye,and the organizational structure and cell morphology were normal by microscope.5.The results of gene chip showed there were 49 GO categories with significant difference of gene expression between between normal hepatocytes and MSCs;and only 15 GO categories related to cell growth and biological regulation had significant difference of gene expression between normal hepatocytes and hepatocytes after differentiation.【Conclusion】1.MSCs can be isolated and cultured from rat bone marrow by adherent culture screening method and density gradient centrifugation.MSCs by density gradient centrifugation cultured were more pure.Rat bone marrow MSCs strongly expressed CD29,CD90,and not to express CD34.2.Cell factor(SCF,HGF,FGF-4) and hepatic extract fluid could induce MSCs to differentiate into hepatocytes,while hepatic extract fluid had the best induced results.3.Detected the albumin and urea production in induced culture supernatant,the expression of CK18 and the intake of ICG could comprehensively identify differential cells was the hepatocytes.4.Hepatocytes after differentiation induced by common cytokines(SCF,HGF,FGF-4) and hepatic extract fluid had no tumor formation in the nude mice.5.At the gene level,there were significantly differences in gene expression between rat normal hepatocytes and MSCs.However,there were no significantly differences in gene expression between rat normal hepatocytes and hepatocytes induced by the hepatic extract fluid.Hepatocytes after differentiation were similar to normal hepatocytes in the cell function,metabolism,and regulation of enzyme activity.