体外构建血管内皮生长因子短发夹状RNA表达载体及其对人骨肉瘤细胞相应因子mRNA及蛋白的抑制

Inhibition of the expression of vascular endothelial growth factor in osteosarcoma cells by a short hairpin RNA

目的:设计并构建血管内皮生长因子短发夹状RNA表达载体,检测其对人骨肉瘤细胞血管内皮生长因子mRNA及蛋白表达的影响。方法:实验于2006-01/07在青岛市市立医院试验中心完成。人骨肉瘤细胞系MG-63购自武汉大学生命科学院;pSilence2.1-neo载体带有U6启动子和neo基因(Ambin公司)。①以人血管内皮生长因子的mRNA序列5’-GATAACGCGGATACCTTGG-3’为靶点,体外构建血管内皮生长因子短发夹状RNA表达载体pSilence2.1-neo-VEGF。②人骨肉瘤细胞MG-63常规培养,消化传代后分为3组:短发夹状RNA载体组转染pSilence2.1-neo-VEGF载体,空载体组转染pSilence2.1-neo载体,空白组无特殊处理。③转染后48h收集各组细胞,采用RT-PCR检测血管内皮生长因子mRNA的表达,免疫组化法检测血管内皮生长因子蛋白的表达。结果:①各组细胞血管内皮生长因子mRNA的表达情况:短发夹状RNA载体组VEGF165mRNA的相对含量显著低于空白组、空载体组(0.181±0.010,1.064±0.019,1.052±0.036,P<0.01),而空白组和空载体组之间差异无显著性意义。②各组细胞血管内皮生长因子蛋白的表达:空白组、空载体组多数细胞中出现明显的棕黄色颗粒状细胞质着色,而短发夹状RNA载体组细胞着色较淡。短发夹状RNA载体组的阳性细胞率显著低于空白组、空载体组[(20.12±2.63)%,(92.45±7.56)%,(91.76±5.26)%,P<0.01],而空白组和空载体组之间差异无显著性意义。结论:pSilence2.1-neo-VEGF可显著抑制人骨肉瘤细胞MG-63血管内皮生长因子mRNA及蛋白的表达,为靶向血管的骨肉瘤基因治疗提供了参考依据。

AIM： To construct an expression plasmid of a short hairpin RNA targeting vascular endothelial growth factor （VEGF）, and investigate its inhibitory effect on the expression of VEGF in osteosarcoma cell line MG-63 in vitro. METHODS： The experiment was completed in the experimental center of Qingdao Municipal Hospital from January to July in 2006. Human osteosarcoma cell line MG-63 was purchased from College of Life Sciences in Wuhan University, pSilence2.1-neo vector had U6 promoter and neo gene （Ambin Corporation）①An expression plasmid pSilence2.1-neo-VEGF of a short hairpin RNA was construct in vitro targeting VEGF mRNA series 5＇-GAT AAC GCG GAT ACC TYG G-3＇.②The MG-63 cells were cultured under regular condition and then divided into three groups： control group without treatment, blank plasmid group transfected with pSilence2.1-neo and transfected group with pSilence2.1-neo-VEGF165. ③ Cells were collected 48 hours after transfection and the expression of VEGF mRNA and protein in three groups were analyzed by RT-PCR and immunohistochemical methods respectively.. RESULTS： ①The expression of VEGF mRNA in three groups： The expression of VEGF165 mRNA in the transfected group was significantly lower than that of other two groups （0.181 ±0.010, 1.064 ±0.019, 1.052±0.036, P 〈 0.01）, but there was no significant difference between the control group and the blank plasmid group. ② The expression of VEGF protein in three groups： There were obviously deep yellow stain in the control group and the blank plasmid group, but the stain in the transfected group was weaker. The positive cell value of the transfected group was significantly lower than that of other two groups [（20.12±2.63）%, （92.45±7.56）%, （91.76±5.26）%, P 〈 0101]. There was no significant difference between the control group and the blank plasmid group. CONCLUSION： pSilence2.1-neo-VEGF can significantly reduce the expressions of VEGF mRNA and protein in osteosareoma cell line MG-63, which provides the references for the gene theranv of osteosarcoma.