反义基因转染抑制骨肉瘤中血管内皮生长因子表达的研究

Inhibition of vascular endothelial growth factor expression in osteosarcoma transfected by Adeno-associated virus gene

目的 观察以腺相关病毒 (rAAV)为载体的血管内皮生长因子 (VEGF)反义基因转染的骨肉瘤细胞中VEGF蛋白的表达。方法 用不同量 (0、2 0、5 0、10 0、2 0 0、2 40 μl)的以rAAV为载体包装的反义VEGF基因 (rAAV antiVEGF)转染人骨肉瘤细胞系MG 63细胞 ,收集转染前后的培养细胞及上清 ,用链霉素抗生素蛋白 过氧化酶连接法 (SP)和免疫印迹法 (Western blot)检测VEGF蛋白的表达 ;用逆转录聚合酶链式反应 (PCR)检测VEGFmRNA表达。结果 SP结果表明转染rAAV antiVEGF基因的量越多 ,显色越淡 ;Western Blot结果表明转染rAAV antiVEGF基因的量越多 ,其灰度越低。测得不同量 (0、2 0、5 0、10 0、2 0 0、2 40 μl)的转染基因其相应的吸光度值分别为 86614± 13 776、73 2 45± 15 414、610 78± 12 12 4、5 465 7± 10 95 3、3 980 2± 113 0 8、3 2 0 14±15 0 5 7,差异有显著性 (P <0 .0 5 ) ;PCR检测其灰度值分别为 0 .986、0 .913、0 .784、0 .60 6、0 .43 1、0 .40 8,差异有显著性 (P <0 .0 5 )。结论 rAAV antiVEGF可封闭VEGFmRNA ,使已转染rAAV antiVEGF的MG 63细胞表达的VEGF蛋白减少。

Objective To evaluate the expression of vascular endothelial growth factor (VEGF) protein in osteosarcoma cells transfected by adeno-associated virus (rAAV)-antisense VEGF.Methods Different doses of rAAV-antisense VEGF (0,20,50,100,200,240 μl) were transfected into osteosarcoma MG-63 cells.The cells and culture supernatant before and after transfection were collected.The expression of VEGF was detected by immunohistochemical staining (SP) and Western blot;The expression of VEGF mRNA was detected by polymerase chain reaction (PCR).Results SP and Western-blot assays revealed that the rAAV-antisense VEGF gene-transfected MG-63 cells had weaker staining than those without transfected by rAAV-antisense VEGF-gene,and staining intensity was negatively related with doses.The cells transfected by different doses of gene (0,20,50,100,200,240 μl) had different absorbance:86 614±13 776,73 245±15 414,61 078±12 124,54 657±10 953,39 802±11 308,32 014±15 057,with the difference being significant by Chi-square test ( P <0.05).The expression of VEGF mRNA had the similar results and its absorbance was 0.986,0.913,0.784,0.606,0.431,0.408 respectively ( P <0.05).Conclusion The expression of VEGF protein in MG-63 cells can be inhibited by rAAV-antisense VEGF.