Objective To solve the shortage of hepatocytes for l iver tissue engineering, to explore the possibil ity of prol iferation of rat bone marrow mesenchymal stem cells (BMSCs) and the feasibil ity of differentiation of BMSCs into hepatocyteswith a culture system containing cholestatic rat serum and hepatocyte growth factor (HGF) in vitro. Methods Myeloid cellsof femur and tibia were collected from the female healthy Wistar rats at the age of 6 weeks, the BMSCs were isolated, purified and identified. Normal and cholestatic rat serum were prepared from 40 healthy Wistar rats at the age of 12-14 weeks. The 3rd passage of BMSCs were harvested and added different cultures according to the following grouping: group A, DMEM plus 10%FBS; group B, hepatocyte growth medium (HGM) plus 5%FBS; group C, HGM plus 5% normal rat serum; group D, HGM plus 5% cholestatic rat serum; group E, HGM plus 5% cholestatic rat serum plus 25 μg/L HGF. The changes of cell morphology were observed, MTT assay was used to measure cell growth; the expression of alpha-fetoprotein (AFP) and cytokeratin 18 (CK18) were detected by immunocytochemistry; the glycogen deposit was examined by periodic acid-schiff (PAS) staining; and the urea content in culture supernatant was determined by glutamate dehydrogenase. Results Polygonal cells and binuclear cells were observed in groups D and E, while the shapes of cells in groups A, B, and C did not obviously change. The cell growth curve demonstrated that the speed of cells proliferation in group C was the fastest, the one in group B was the slowest; showing significant differences when compared with groups A, D, and E (P lt; 0.05). On the 7th day in groups D and E, the positive expressions of AFP and CK18 emerged, on the 14th day the positive expression of glycogen emerged. At the same period, the expression ratio was higherin group E than in group D (P lt; 0.05). The urea concentration increased gradually with induction time in groups D and E, the concentration was higher in group E than in group D (P lt; 0.05). No expressions of AFP, CK18, glycogen, and change of the urea concentration were observed in groups A, B, and C. Conclusion Normal rat serum can obviously promote the growth of BMSCs; cholestatic rat serum which promote the growth of BMSCs can induce to differentiate into hepatocyte; and a combination of cholestatic serum and HGF can increase the differentiation ratio.
Objective To investigate the effect of hepatocyte-l ike cells induced by CD34+ cells in vitro on the repair of the injured hepatic tissues of mice in vivo. Methods Mononuclear cells were isolated from umbil ical blood by density gradient centrifugation and enriched CD34+ cells were obtained. The cells were (1 × 105 cells/mL) cultured in serumfreemedium containing stem cell factor (SCF), hepatocyte growth factor (HGF), EGF, oncostatin M (OSM), bFGF (the concentration were 50, 20, 20, 10, 10 ng/mL respectively) in vitro for 10 days. Forty-eight 6-week-old female ICR mice werechosen to prepare l iver injury model by injecting carbon tetrachloride and 2-acetylamionoflu-orene. The mice were randomly divided into two groups (n=24 per group): the experimental group, the cultured cells were injected into the mice through the tail vein; the control group, the equivalent serum-free medium was injected. Six mice from each group were killed at 7, 14, 21, and 28 days after operation to receive HE staining, PCR gel electrophoresis, immunohistochemistry staining, and hepatic function detection. Results HE staining: the morphology of injured hepatic tissues in the control group recovered to normal 28 days after operation, while in the experimental group, it recovered to normal 14 days after operation. PCR gel electrophoresis and immunohistochemistry staining: the cells expressing human serum albumin were detected in the hepatic tissue of the experimental group at each time point after operation; while in the control group, no such cells were detected within 28 days after operation. Hepatic function detection: the activity of alanine aminitransperase in the control group recovered to normal 14 days after operation; the mean activity of aspartate aminotransferase of two groups failed to recover within 28 days. Conclusion The hepatocyte-l ike cells induced by CD34+ cells in vitro can promote the morphological and functional recovery of the injured hepatic tissue in mice. Moreover, it can be transformed into human-derived hepatic cells in l iver-injured mice.