Abstract:

Abstract:Objective
To study the effects of TRAIL and endostatin double genes in recombinant plasmid pshuttle-Egr1-shTRAIL-shES combined with X-ray irradiation on the proliferation,cell cycle procession and apoptosis in human vascular endothelial ECV304 cells.Methods Cells were divided into control,empty vector pshuttle,TRAIL single-gene plasmid pshuttle-Egr1-shTRAIL,endostatin single-gene plasmid pshuttle-Egr1-shES and TRAIL plus endostatin double-gene plasmid pshuttle-Egr1-shTRAIL-shES transfection groups.The cell transfection was done by lipofectamine-mediated method,and no transfection in control group.After plasmid transfection,the cells were exposed under X-ray irradiation at the doses of 0,0.1,0.5,1.0,2.0 and 5.0 Gy,  respectively.Then the time-course and dose-effect patterns of the TRAIL and endostatin protein expressions induced by irradiation were detected by ELISA,and the effects of TRAIL and/or endostatin single or double gene therapy combined with radiotherapy on the proliferation,cell cycle procession and apoptosis in ECV304 cells were detected by MTT assay,flow cytometry (FCM) with PI single-staining or/and Annexin Ⅴ double-staining.Results After 2.0 Gy X-ray irradiation,the expression levels of TRAIL and endostatin proteins in the supernatant of cultured ECV304 cells transfected with pshuttle-Egr1-shTRAIL-shES at other time-points were increased when compared with those at 0 h (P<0.01),and reached to the peak value at 24 and 12 h, respectively.Furthermore,their expression levels of TRAIL and endostatin protein induced by irradiation were increased significantly with the enlargement of radiation doses (P<0.05 or P<0.01).Under the exposure of X-ray irradiation,as compared with those in  control and pshuttle groups,the A490 values detected with MTT in pshuttle-Egr1-shTRAIL,pshuttle-Egr1-shES and pshuttle-Egr1-shTRAIL-shES groups were reduced in a time- and dose-dependent manner,and the apoptotic rates and the cell percentages in G^₂ + M phase were increased,while those in G₀/G₁ phase were declined.In particular,the cellular effects mentioned above in pshuttle-Egr1-shTRAIL-shES group were significantly higher than those in pshuttle-Egr1-shTRAIL and pshuttle-Egr1-shES groups(P<0.05 or P<0.01).Conclusion TRAIL and endostatin double-gene-therapy in combination with radiotherapy could inhibit cell proliferation,affect cell cycle procession and promote apoptosis in ECV304 cells,and its therapeutic efficacy is better than those of single gene-radiotherapy or radiotherapy.

Key words: endostatin, early growth response-1 promoter, gene-radiotherapy, tumor necrosis factor related apoptosis inducing ligand