Inhibitory effect of vascular endothelial growth factor-antisense oligonucleotide on the proliferation of vascular endothelial cells in human cerebral arteriovenous malformation / 中国组织工程研究

ABSTRACT

BACKGROUND:Antisense gene therapy offers immense promise for the management of human cerebral arteriovenous malformation through inhibiting expression of vascular endothelial growth factor and angiogenesis in endothelial cells.OBJECTIVE: To observe the inhibitory effect of vascular endothelial growth factor-antisense oligonucleotide (VEGF-ASODN) on the proliferation of vascular endothelial cells in human cerebral arteriovenous malformation.DESIGN: Observational contrast study.SETTING: Department of Neurosurgery, General Hospital of Shenyang Military Area Command of Chinese PLA.MATERIALS The experiment was carried out in the Neuromedical Institute, General Hospital of Shenyang Military Area Command of Chinese PLA from August to December 2006. A total of 18 patients with human cerebral arteriovenous malformation were selected from Department of Neurosurgery, Shenyang General Hospital of Military Area Command of Chinese PLA. There were 12 males and 6 females and their mean age was 40 years. Cerebral arteriovenous malformation was classified based on Spetzler grade grade Ⅱ (n =10) and grade Ⅲ (n=8). All cases were diagnosed with whole cerebral angiography before operation and they provided the confirmed consent. Main reagents were detailed as follows endothelial cell growth supplements (ECGS, Sigma, USA), 391 DNA automatic synthetic device (Shanghai Shenggong Liyong Company, PE, USA), anaerobic incubator (DY-1, Zhejiang), human vascular endothelial growth factor enzyme-linked kit (TBD Company, Beijing), 96E enzyme-labeling device (ERMA, INC), cell cycle analytical reagent kit (BD Company), and flow cytometer (FACS Calibur, BD Company).METHODS: ①Experimental procedure Tissue explants adherent method was used to culture vascular endothelial cells from human cerebral arteriovenous malformation. The third generated cells were used and randomly divided into antisense group, sense group and control group with four bottles of cells in each group. Sense and antisense phosphorothioate oligodeoxynucleotides of artificial vascular endothelial growth factor selected from the antisense group and the sense group were covered with positive liposomes, and then they were used to transfected vascular endothelial cells cultured from human cerebral arteriovenous malformation; however, cells in the control group were not dealt with any treatments. Cells in the three groups were incubated in anaerobic incubator (including 0.95 volume fraction of N2 and 0.05 volume fraction of CO2) at 37 ℃ for 2, 4 and 8 hours, respectively. ② Experimental evaluation Cell cycles were measured, protein content of vascular endothelial growth factor was measured, and mRNA expression of vascular endothelial growth factor was also detected.MAIN OUTCOME MEASURES: Expression of mRNA and protein of vascular endothelial growth factor and proliferation exponent at different times of hypoxia.RESULTS: ①mRNA expression of vascular endothelial growth factor At 2, 4 and 8 hours after hypoxia, mRNA expression of vascular endothelial growth factor was higher than that before hypoxia in the control group (P ＜ 0.05);however, mRNA expression was lower in the antisense group than that in the control group (P ＜ 0.05). ② Protein content of vascular endothelial growth factor At 2, 4 and 8 hours after hypoxia, protein content of vascular endothelial growth factor was higher than that before hypoxia in the control group (P ＜ 0.05); however, protein content was lower in the antisense group than that in the control group (P ＜ 0.05). ③ Proliferation exponent At 4 and 8 hours after hypoxia,proliferation exponent of endothelial cells cultured from human cerebral arteriovenous malformation was higher than that before hypoxia in the control group (P ＜ 0.05); however, proliferation exponent was lower in the antisense group than that in the control group (P ＜ 0.05).CONCLUSION: Hypoxia may induce gene expression of vascular endothelial growth factor in endothelial cells at the transcriptional level. Antisense vascular endothelial growth factor can obviously inhibit gene expression of vascular endothelial growth factor cultured from human cerebral arteriovenous malformation and proliferation under hypoxic conditions.