RESUMO
BACKGROUND:Umbilical cord mesenchymal stem cells can be induced to differentiate into hepatocyte-like cells in vitro and in vivo.However,the exact mechanism is still unknown. Existing studies have shown that the Wnt/β-catenin signaling pathway is closely related to this process. OBJECTIVE: To explore the effect of Wnt/β-catenin signaling pathway on the differentiation of umbilical cord mesenchymal stem cells into hepatocyte-like cells and its potential molecular mechanism. METHODS: Human umbilical cord mesenchymal stem cells were extracted from the neonatal umbilical cord by tissue adherent method. After being cultured and purified, the umbilical cord mesenchymal stem cells at passages 4-6 were divided into four groups: control group (DMEM culture group), hepatocyte-like differentiation group, activator Wnt3a group (adding 20 μg/L Wnt3a, an activator of Wnt/β-catenin signaling pathway, under the differentiation condition), and inhibitor Dkk-1 group (adding 20 μg/L Dkk-1, an inhibitor of Wnt/β-catenin signaling pathway, under the differentiation condition). Induced cells were collected respectively on days 7, 14, 21, 28. Their mRNA and protein expressions of α-fetoprotein (AFP), albumin (ALB), hepatocyte nuclear factor 4α (HNF4α) and Cytokeratin-19 (CK-19) in the cells were detected by real-time quantitative PCR and western blot respectively. Meanwhile, Periodic Acid-Schiff staining, low-density lipoprotein uptake test and indocyanine green absorption test were applied to detect the function of hepatocyte-like cells. RESULTS AND CONCLUSION: Compared with the control group, expressions of AFP and HNF4α mRNA and protein as well as ALB mRNA were significantly up-regulated in the hepatocyte-like differentiation group, activator Wnt3a group and inhibitor Dkk-1 group (P < 0.05). Whereas, there was a decrease in the CK-19 expression at mRNA and protein levels (P < 0.01) in these three groups. Compared with the hepatocyte-like differentiation group, the mRNA and protein expressions of AFP and HNF4α, and the mRNA expression of ALB were significantly down-regulated in the activator Wnt3a group (P < 0.05). Compared with hepatocyte-like differentiation group and activator Wnt3a group, the inhibitor Dkk-1 group had higher expression of AFP, HNF4α mRNA and their proteins as well as the mRNA expression of ALB (P <0.05). Findings from the Periodic Acid-Schiff staining, low-density lipoprotein uptake test and indocyanine green absorption test showed more positive cells in the inhibitor Dkk-1 group than in the hepatocyte-like differentiation group and least positive cells in the activator Wnt3a group. Overall, these findings suggest that the inhibition of Wnt/β-catenin signaling pathway promotes the differentiation of umbilical cord mesenchymal stem cells into hepatocyte-like cells;conversely,the cell differentiation can be inhibited via the Wnt/β-catenin pathway.
RESUMO
Objective: To study the extracting technology of Antrodiacamphorata total triterpenoids (ACTT) and their anti-tumor activity. Methods: To optimize the ACTT extraction process by central composite design of response surface methodology (CCD-RSM).To compare the inhibition of ACTT extract at different concentration and different time on in vitroproliferation of cultured HepG2 cell using MTT assay;To observe the HepG2 cell morphology by fluorescence microscopy and flow cytometry andto detect the cell apoptosis rate by AnnexinV apoptosis assay kit. Results:The optimized process was plus 20 times amount of 90% ethanol, ultrasonic extraction twice, 40min each time, ultrasonic power of 400W.ACTT extracting rate was 11.87%, deviation between the actual and predicted values of ACTT extraction rate was 1.56%.MTT assay showed that in 12.5-200μg/mL, ACTT extract at different concentration (12.5-200μg/mL) on the proliferation of HepG2 cells showed significant inhibition(P<0.01).Microscopic observation and direct form Hoechst33342 staining were used to observethenuclear enrichment and nuclear fragmentation and other typical features of apoptosis and apoptotic bodies;Flow cytometry showed that there was significant differenceof apoptosis rate in the administration group (P<0.01) compared with the control group. Conclusion: UsingCCD-RSM to optimize antrodia ultrasonic alcohol extraction process, theactual and predicted values are consistent with high and good predictability which is feasible.ACTT extract can significantly inhibit the proliferation and induce apoptosis on HepG2 cells in vitro.