Thymosin β4Promotes Differentiation Of Hepatic Progenitor Cells Into Hepatocytes In Vitro

Objective:To explore the proliferation and differentiation effects of thymosin β4(Tβ4) on hepatic progenitor cells(WB-F344cells), and to study the functions of the induced cells by Tβ4, thus to provide the theoretical basis for Tβ4in promoting hepatic progenitor cells differentiation and the liver regeneration.Methods:1. WB-F344cells were cultured with same amount of medium contained various concentrations of T(34(1ng/ml,10ng/ml,100ng/ml and1μg/ml), accordingly named lng/ml group, lOng/ml group,100ng/ml group and1μg/ml group, the WB-F344cells cultivated by medium without Tβ4was set as the control group. WB-F344cells’ proliferation was observed by CCK-8assay when cells were cultured for1,2, and3days. The effects of Tβ4on the cells morphology were also observed.2. The groups (10ng/ml,100ng/ml and1μg/ml) were selected of which the cells proliferation effect by Tβ4was statistically significant compared with the control group, and the control group was set at the same time. The gene expression of hepatocyte maker (albumin), liver stem cell maker (alpha-fetoprotein) and cholangiocyte maker (CK-19) were detected by qRT-PCR. Western-blot was used to detect the protein expression of albumin (ALB), alpha-fetoprotein (AFP) and cytokeratin-19(CK-19), thus to confirm what kind of cell was differentiated into. After cells were cultivated for5days, the functions of induced cells was assessed by periodic acid-schiff (PAS) staining and indocyanine green (ICG) up-take assay.Results:1. After the cells cultivated with T04for1and2days, no significant difference in cells proliferation was found when comparing the experimental group with the control group. The proliferation of the cells was slowed down by Tβ4in the groups of10ng/ml,100ng/ml and1μg/ml compared with the control group (P＜0.05). Meanwhile, the induced cells by Tβ4were changed from monocytes into binucleus or multinucleus cells.2. Compared with the control group, the gene expression of ALB was up-regulated when WB-F344cells were incubated with Tβ4for3days in100ng/ml group and1μg/ml group (P＜0.05), the protein expression of CK-19was down-regulated in10ng/ml group,100ng/ml group and1μg/ml group (P＜0.05). when WB-F344cells were incubated with Tβ4for5days, the protein expression of AFP was down-regulated in10ng/ml group,100ng/ml group and1μg/ml group whereas ALB was up-regulated in100ng/ml group and1μg/ml group (P<0.05), and the protein expression of ALB changed in a dose-dependent manner.3. In terms of the functions of induced cells, when cultured with Tβ4for5days, the capability of glycogen storage and the number of positive cells of ICG uptake were higher than the control groups, especially in1μg/ml group.Conclusion:Tβ4promotes differentiation of WB-F344cells into hepatocytes in vitro.The induced cells can exhibit the functions of mature hepatocytes, it provides the theoretical basis for the application of Tβ4on liver regeneration to a certain extent.