The Study On Differentiation Of Hepatocyte Of Rabbit Bone Mesenchymal Stem Cells Induced Through Different Concentration Of HGF And EGF In Vitro

Objective Through study on the different rate of differentiation with different concentrations of hepatocyte growth factor(HGF) and epidermal cell growth factor(EGF) induced rabbit bone mesenchymal stem cells (BMSCs)into hepatocyte in vitro,to find the best combination of concentration of HGF and EGF。Methods BMSCs were isolated,cultured,passaged and purified in vitro by density gradient centrifugation method.Set up the culture system with different concentrations of HGF(30.0ug/L)+EGF(1.5mg/L),HGF(40.0ug/L)+EGF(2.5mg/L),HGF(50.0ug/L)+EGF(3.5mg/L),HGF(60.0ug/L)+EGF(4.5mg/L)。The morphological changes of induced BMSCs were observed in different culture periods.The hepatic surface phenotype including alpha fetoprotein(AFP),albumin(ALB) were identified by immunocytochemistry or immunofluorescent staining at day 7,14,21,28。The concentration of ALB and urea in the supernatant were determined dynamically by bromcresol green and urea assay.Results After 14 days,the fibroblast-like morphology of BMSCs lost and cells became broadened and flattened.After prolonged culture, polygonal cells were seen and further matured,hepatocyte-like morphology were seen by day 21。Immunocytochemistry showed the expression of AFP until day 14,increased on day 21 but reduced in the following days.At vertical up,there were no difference among all groups (P＞0.05).Immunofluorescent staining showed the expression of ALB until day 14,there were no difference in group 1 and group 4 on horizontal(P＞0.05).The positive of ALB increased with the culture time longer in group 2 and group 3(P＜0.05)。on horizontal,there were significantly difference in whole groups,and the higher the concentration, the higher number of positive cells。There were no difference in group 2 and group 3 on day 21,and the same in group 3 and group 4 on day 28 (p＞0.05)。The concentration of ALB in the supernatant were detected on day 12,in the early(9-15 days) showed a concentration-dependent manner, until the peak on the culture day 18 or 21,the concentration decreased,and there were no difference(p＞0.05),The highest value of ALB was at group 4(HGF60ug/L;EGF4.5ug/L)。The concentration of urea were detected on day 9,there were no difference in those groups。Conclusions Rabbit BMSCs are able to differentiate into functional hepatocyte-like cells in vitro with HGF and EGF,just need 2-3 weeks;the rate of differentiation was in a concentration-dependence manner in the early,high concentrations of HGF and EGF may enhance the rate of differentiation.The best combination is HGF 60ug/L and EGF 4.5mg/L.