ABSTRACT
Objective To observe the effect of TGF-β1 on activation and epithelial mesenchymal transition(EMT) in rat hepatic stellate cell-T6.Methods Adopt the MTT method to screen the optimum concentration of TGF-β1 in vitro HSC-T6 cultured.After the HSC-T6 stimulation by TGF-β1 of 10 μg/L for 24 hours, the morphology of the cells was observed under inverted phase contrast microscope, the expression of F-actin which on behalf of cotoskeletal structure was detected by immunofluorescence staining;the expression of α-SMA and N-cadherin,vimentin,E-cadherin was measured by RT-qPCR;The changes of α-SMA,N-cadherin,vimentin and E-cadherin were assessed by Western blot after different concentrations (0,5 and 10 μg/L) of TGF-β1 interventing HSC-T6 for 24 h.Results The optimal cell survival rate was recorded when 10 μg/L TGF-β1 dealt withcells for 24 h.After HSC-T6 were treated with TGF-β1,cells stretched, pseudopodia increased and turn into stellate, cells connections were looser, so that represented a significantly activated state.F-actin filaments gathered to form stress and distributed along the long axis of the cells;The expression of α-SMA mRNA and vimentin mRNA in experimental group was significantly higher while E-cadherin mRNA was obviously lower than the control group(P<0.05).TGF-β1 made the protein expression of α-SMA and N-cadherin, vimentin in dose-dependent increased while E-cadherin was decreased.Conclusions TGF-β1 may induce activation and epithelial-mesenchymal transition of HSC-T6.
ABSTRACT
BACKGROUND:There may be tumor stem cells during metastasis and recurrence of liver cancer.The number of tumor stem cells is very few,so it is difficult to be identified.Thy-1 is a surface marking protein of oval cells.Specific marker can indirectly confirm the existence of tumor stem cells in the tumor,which is a focus in recent studies.OBJECTIVE:To study whether Thy-1 positive marked oval cells was expressed in hepatoma carcinoma cells,and investigate its screening and identification.METHODS:Flow cytometry was used to identify that Thy-1 positive expression in hepatoma carcinoma cells HepG2 and BEL-7402 and normal hepatocytes QSG-7701.Immunomagnetic bead sorting was utilized to select hepatoma carcinoma cells with Thy-1.Immunofluorescence was used to identify selected efficiency.Naked mouse inoculated with hepatoma carcinoma cells in the skin test was assigned to three groups.In the Thy-1~+ cells group,naked mice were inoculated with Thy-1~+ cells (5×10~5/mouse)underneath the skin of the back.In the HepG2 cell positive control group,naked mice were inoculated with HepG2 cells(0.5×10~7/mouse)underneath the skin of the back.In the Thy-1' cells negative control group,naked mice were inoculated with Thy-1~- cells(0.5×10~7/mouse)underneath the skin of the back.Tumorigenicity in each group was observed one month later.RESULTS AND CONCLUSION:Thy-1 expression in hepatoma carcinoma cells HepG2 and BEL7402 was significantly greater compared with normal hepatocytes QSG7701(P＜ 0.05).Positive rate of immunofluorescence staining was 85% in the Thy-1~+ cell group,1% in the HepG2 cell positive control group,0% in the Thy-1~- cell negative control group.There were nubble of five mice in HepG2 cell positive control and Thy-1~+ groups,and the volume was large.However,there was one nubble in Thy-1~- group,and the volume was significantly small(F=144.568,P＜ 0.05).Results indicated that hepatoma carcinoma cells contained Thy-1 labeled positive oval cells-like stem cells,which may be tumor stem cells in hepatoma carcinoma cells.