The Establishment Of Human Venous Malformation Cell Model And The Experimental Study On The Mechanism Of Pingyangmycin

Objective:A venous malformation cell model is established to observe the effect of pingyangmycin on the proliferation and apoptosis of venous malformation cells,and to explore the potential mechanism of pingyangmycin-induced apoptosis of venous malformation cells.Methods:1.Construction of venous malformation cell model and wild type cell model:TIE2-L914F and TIE2-WT genes were overexpressed in human umbilical vein endothelial cell(HUVEC)to construct a venous malformation cell model and a wild type cell model by lentiviral transfection.Puromycin was used to do a resistance screening.HUVEC was a control group.2.Identification of venous malformation cell model and wild type cell model:After 72 hours of lenti virus transfection,the expression of green fluorescent protein(GFP)in HUVEC was observed by inverted fluorescence microscope,and the transfection efficiency of TIE2-L914F and TIE2-WT groups was calculated.QRT-PCR was used to detect the expression level of TIE2 gene in each group,and Western blot was used to detect the expression level of TIE2 and p-TIE2 protein in each group.3.CCK-8 was used to detect the effect of pingyangmycin on the proliferation of three groups of cells:Three groups of cells in logarithmic growth period were inoculated into 96 well plate according to 1 × 104 cells/well.Four parallel holes and a blank group(PYM concentration:lug/ml,l0ug/ml,50ug/ml,100ug/ml,200ug/ml)were set in each group for 24 hours and 48 hours respectively.The cell survival rate(cell proliferation activity)of each group was calculated.4.Hoechst 33342 staining was used to detect the effect of pingyangmycin on the apoptosis of three groups of cells:Three groups of cells in logarithmic growth period were inoculated into 12 well plate according to 1 X 105 cells/well.Each group was set treatment groups and blank group(without PYM treatment).Hoechst 33342 was used to stain and fluorescence microscope was used to observe and photograph.5.Flow cytometry was used to detect the effect of pingyangmycin on the apoptosis rate of three groups of cells:Three groups of cells in logarithmic growth period were inoculated into 6 well plate according to 2×105 cells/well.Each group was set treatment groups and blank group(without PYM treatment).Cells in each group were collected and stained with FITC/PI.Flow cytometry was used to detect the proportion of apoptosis in each group.6.Western blot was used to detect the expression of apoptosis-related proteins in three groups of cells:Three groups of cells in logarithmic growth period were inoculated into 6 well plate at 2 X 105 cells/well.Each group was set treatment groups and blank group(without PYM treatment).The total protein of each group was collected.Western blot was used to detect the expression level of apoptosis-related protein p53?Bax?Bcl-2?Caspase-3?Caspase-9 in each group.Results:1.The venous malformation cell model and wild type cell model were successfully established in vitro.The GFP fluorescence intensity and number of cells in both groups were higher(transfection rate>80%),and the expression level of TIE2 mRNA and p-TIE2 protein were more significantly higher than those in control group,the p-TIE2 protein level in TIE2-L914F group was significantly higher than that in TIE2-WT group.2.CCK-8 results showed that pingyangmycin can significantly inhibit the proliferation of venous malformation cells in a time-and dose-dependent manner.At the same dose and time,pingyangmycin significantly inhibited the proliferation of venous malformation cells than the other two groups.Transfection of lentivirus alone had no effect on the proliferation activity of HUVEC cells.3.Hoechst 33342 fluorescence staining showed that the number of apoptotic bodies in TIE2-L914F group increased significantly compared with TIE2-WT group and control group.Transfection of lentivirus alone had no obvious effect on the apoptosis of HUVEC.4.Flow cytometry results showed that the proportion of apoptotic cells in TIE2-L914F group increased significantly compared with TIE2-WT group and control group.Transfection of lentivirus alone had no obvious effect on the apoptosis of HUVEC.5.Western blot results showed that the expression level of p53 and Bax pro-apoptosis-related proteins in TIE2-L914F group were up-regulated;the activation level of Caspase-3 and Caspase-9 pro-apoptosis-related proteins were up-regulated;the expression level of apoptosis inhibitory protein Bcl-2 was down-regulated.Transfection with lentivirus alone had no effect on the expression of apoptosis-related proteins in HUVEC.Conclusion:Overexpression of TIE2-L914F in HUVEC to establish a venous malformation cell model is feasible which lays a foundation for experimental research of venous malformation;Pingyangmycin can significantly inhibit the proliferation of venous malformation cells and may promote apoptosis through mitochondrial pathway,thus playing a therapeutic role.