Rat Bone Marrow Mesenchymal Stem Cells Differentiatied Into Hepatocyte-like Cells And Treatment Of Acute Liver Failure

Objectives: (1) To explore the induction method of the rat bone marrow mesenchymal stem cells into hepatocyte-1ike cells and observe the differentiation ability of BMSCs. (2) To explore the treatment of bone marrow mesenchymal stem cells to rats with acute liver failure.Methods: (1) BMSCs of SD rats was purified by the method of whole bone marrow attachment culture, digested by trypsin and then passaged. At the third passage, BMSCs were differentiated into hepatocyte-like cells by Hepatocyte growth factor(HGF) and Fibroblast growth factor-4(FGF-4) in vitro. Cells were collected on day 7th,14th,21st, the morphological changes of cells and the expression of liver cells markers were observed. The expression of albumin(ALB),alpha-fetoprotein(AFP) and cytokeratin18(CK18) mRNA was detected by reverse transcription-polymerase chain reaction(RT-PCR). Glycogen was tested by periodic acid-schiff (PAS) on days 7,14 and 21 after induction. The expression of albumin detected by immunofluorescence on day 14,21, to verify the results of induced differentiation. (2) A rat model of acute liver failure by injecting CCl4 was established. After 24 hours, 1ml DMEM/F12 suspension containing 2.0×106 BMSCs cells which was induced and non-induced were injected into the same species rats via the tail vein. Rats in the injured liver saline group were infused with anequal volume of saline. To observe the diet, activity and death situation of the rats, and all rats were sacrificed to obtain liver and serum samples at 3,7 and 14 days following transplantation.Results: (1) With the cultured time longer, BMSCs showed the features of hepatocyte-like, round cells appeared and showed colony growth, liver cell-specific markers began to emerge and mature. RT-PCR showed that the differentiated cells expressed AFP from day 7, and increased significantly on day 14, then decreased from day 21. Cells expressed CK18, ALB and glycogen on day 14, and boosted gradually during the maturation process. The expression of albumin in the cytoplasm is positive by detection of immunofluorescence on day 14 after induction, and increased significantly on day 21. (2) Different doses of CCl4 may lead to different degrees of liver damage, while the doses of 50% CCl4 4ml/kg simulate the pathophysiology of acute liver failure to change a good stability. After transplantation for 3～14d, two cells transplant groups could be detected the positive cells labled in DAPI in the liver. The recovery of the transplantation group was obviously better than the control group, and the induction group was slightly better than non-induced group.Conclusion: (1) Bone marrow mesenchymal stem cells have the ability of differentiating into hepatocyte-like cells by the method of whole bone marrow attachment culture, HGF,FGF-4 and other factors in vitro. (2) BMSCs were transplantated through tail vein is effective for the recovery of hepatic function in the ALF rats.