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1.
Chinese Journal of Tissue Engineering Research ; (53): 6517-6520, 2007.
Article in Chinese | WPRIM | ID: wpr-407825

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.

2.
Chinese Journal of Tissue Engineering Research ; (53): 7278-7281, 2007.
Article in Chinese | WPRIM | ID: wpr-407686

ABSTRACT

BACKGROUND:Shear stress can directly mediate the expression of endothelial cells, especially some cytokine genes whose codes are related to angiogenesis. Otherwise, flow shear stress of blood plays an importantly biological role in regulating vascular structure and function.OBJECTIVE: To observe the effects of laminar flow shear stress on the proliferation of vascular endothelial cells and the expression of protooncogene c-myc in human cerebral arteriovenous malformation.DESIGN: Randomized controlled study.SETTING: Department of Neurosurgery, Shenyang General Hospital of 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 November 2006 to February 2007. Fresh samples of human cerebral arteriovenous malformation were derived from 20 patients who were of grade Spetzler Ⅱ -Ⅲ and received resection of human cerebral arteriovenous malformation in the Department of Neurosurgery, General Hospital of Shenyang Military Area Command of Chinese PLA in 2006. All cases were diagnosed with whole-brain angiography before operation. The main reagents and equipments were detailed as follows: M199 culture media (Gilbco BRL), quality fetal bovine serum (HyClone), endothelial cell growth supplement (ECGS; Sigma, USA), CO2 incubator (Forma Scientific, USA), flow cytometry analysis of cell cycle kit (BD Company), flow cytometer (FACS Calibur, BD Company), rat-anti-human c-myc monoclonal antibody (Santa Cruz Company, USA), and reverse transcription polymerase chain reaction (RT-PCR) kit (Promega).METHODS: Tissue explants adherent method was used to culture vascular endothelial cells of human cerebral arteriovenous malformation, and then the cells were classified into 4 groups based on degree of shear stress, including control group, low shear stress group, moderate shear stress group and high shear stress group. Cultured endothelial cells of human cerebral arteriovenous malformation were put in a parallel plate flow chamber. In addition, cells in the low,moderate and high shear stress groups were stressed by low, moderate and high shear stress for 8 hours, respectively.However, shear stress in the control group was 0 Pa. Flow cytometry was used to measure proliferation index, and the expression of c-myc protein and c-myc mRNA were determined by immnocytochemistry and RT-PCR analysis respectively.MAIN OUTCOME MEASURES: Expressions of c-myc protein and c-myc mRNA and proliferation index in endothelial cells under various degrees of shear stress.RESULTS: ① Proliferation index: Proliferation index was higher in the moderate and high shear stress groups than that in the control group (P < 0.05, 0.01). ② Expression of c-myc protein: Immuneposjtjve expression of c-myc protein was gradually increased with the increase of shear stress and there were significant differences in the three shear stress groups as compared with control group (P < 0.05-0.01). ③ Expression of c-myc mRNA: Proliferation index of endothelial cells was higher in the low and moderate shear stress groups than that in the control group (P < 0.05).CONCLUSION: Flow shear stress can induce expression of c-myc and activate expression of c-myc gene based on gene transcription so as to promote the proliferation of vascular endothelial cells in human cerebral arteriovenous malformation

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