| Objective: The aim of this study was to investigate the role of HGF in inhibiting endothelial-mesenchymal transition(End MT)of human vascular endothelial cells(HUVECs)induced by TGF-β1,and the possible role of Notch signaling pathway in this process,and to look for possible inhibition of End MT and possible therapeutic targets for diseases caused by End MT.Methods :(1)Establishment of endothelial-mesenchymal transition model induced by TGF-β1 in human umbilical vein endothelial cells: Different concentrations of human TGF-β1 protein were applied to human vascular endothelial cells for 72 hours.The morphological changes of the cells were observed under an inverted microscope after the action.The total RNA was extracted and then the m RNA expression levels of endothelial cell marker(CD31)and mesenchymal cell markers(α-SMA,FSP1)were detected by RT-PCR.After the comprehensive analysis,human TGF-β1 protein at a concentration of 10ng/ml was used for subsequent experiments.(2)Modeling of c-Met gene silencing of HUVECs by c-Met si RNA: HUVECs were transfected with fluorescent labeled si RNA at different concentrations for 6 hours.The transfection efficiency of each concentration of si RNA was observed under a fluorescence microscope,and subsequent experiments were carried out using 50 n M si RNA.Then HUVECs were transfected with different fragments of si RNA(001,002,003).After transfection,the expression of c-Met m RNA was detected by RT-PCR.The si RNA of 001 fragment was used for subsequent experiments.(3)Method for loading each group of experiments: The experiment was divided into 4 groups: blank control group(Ctr group),TGF-β1 group(T group),TGF-β1+HGF group(T+H group)and TGF-β1 HGF c-Met si RNA group(T+H+C/S group).HUVECs were inoculated into 6-well plates,and then cultured in complete medium to a cell density of 30-40%.TGF-β1+HGF+c-Met si RNA group cells were transfected by c-Met si RNA for 6 hours.When the cell density reached 60-70%,the medium were replaced with the growth factor-free medium,and appropriate amount of human TGF-β1 protein and human HGF protein were added in groups to a final concentration of 10ng/ml,and then cultured in a 37 ° C,5% CO 2 incubator for 72 hours.Morphological changes of each group were observed under an inverted microscope at 24,48 and 72 hours.(4)Real-time PCR: Establishe relevant models according to the requirements of each group and complete the relevant processing of cells,then extracted total cellular RNA and measure RNA concentration and purity.After that,reverse-transcribe RNA into c DNA,and detect expression levels of CD31,α-SMA,FSP1,and RBP-J?,in each group by real-time PCR,and the results were statistically analyzed.(5)Western Blotting experiment: According to the requirements of each group,the relevant model establishment and drug intervention were completed,then the total protein of cells was extracted to be measured by BCA method,After the gel making,sample loading,SDS-PAGE electrophoresis,mold transformation,skim milk sealing,primary antibody incubation in 4 refrigerator overnight,and secondary antibody incubation,the spicific bands were imaged by ECL method and were analyzed by Image J software.Results:(1)Establishment of endothelial-mesenchymal transition model induced by TGF-β1 in human umbilical vein endothelial cells: With the increase of TGF-β1 concentration,the expression level of CD31 m RNA decreased gradually,and the m RNA expression levels of α-SMA and FSP1 increased gradually.The above changes were more obvious when the concentration of TGF-β1 was 0-10ng/ml(P< 0.05).As the concentration continued to increase,the trend of each marker was more moderate(P>0.05);24 hours after HUVECs were applied to human TGF-β1 protein at a concentration of 10ng/ml,no obvious morphology was observed under the microscope.Changes,and 48 hours after the action,the cells showed a long spindle-shaped change,and 72 hours after the action,the shape of the cell was long spindle-shaped or spindle-shaped,the morphological changes of cells reached more than 70%.(2)Transfection of HUVECs with c-Met si RNA can effectively inhibit the expression of c-Met gene.(3)The m RNA expression levels of CD31,α-SMA,FSP1 and RBP-J? detected by RT-PCR in each group: the expression of CD31 m RNA in T group was lower than that in Ctr group(P<0.01),and the expression of CD31 m RNA in TH group was higher than that in T group(P<0.01)and T+H+C/S group(P<0.01).The expression of α-SMA m RNA in T group was higher than that in Ctr group(P<0.05).The expression of α-SMA m RNA in T+H group was lower than that in T group(P<0.05)and T+H+C/S group(P<0.01).The expression of FSP1 m RNA in T group was higher than that in Ctr group(P<0.05).The expression of FSP1 m RNA in T+H group was lower than that in T group(P<0.01)and T+H+C/S group(P< 0.01)。The expression of RBP-J? m RNA in T group was higher than that in Ctr group(P<0.05).The expression of RBP-J? m RNA in T+H group was lower than that in T group(P<0.05)and T+H+C/S group(P<0.01).(4)Detection of CD31,α-SMA,RBP-J? protein expression by Western Blotting: The expression of CD31 protein in T group was lower than that in Ctr group(P<0.01).The expression of CD31 protein in T+H group was higher than that in T group(P<0.01)and T+H+C /S group(P<0.01).The expression of α-SMA protein in T group was higher than that in Ctr group(P<0.01),and the expression of α-SMA protein in T+H group was lower than that in T group(P<0.01)and T+H+C/S group(P<0.01).The expression of RBP-J? protein in T group was higher than that in Ctr group(P<0.01).The expression of RBP-J? protein in T+H group was lower than that in T group(P<0.01)and T+H+C/S group(P<0.01).Conclusions:(1)TGF-β1 can induce End MT in HUVECs.(2)HGF can inhibit TGF-β1-induced End MT.(3)Activation of Notch signaling pathway is involved in the process of End MT of HUVECs.HGF can inhibit End MT by down-regulating the Notch signaling.(4)This experiment provides a possible direction for the treatment of End MT-associated vascular diseases. |
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