RNA干扰真核表达载体pEGFP-H1介导的血管内皮生长因子shRNA治疗人胶质瘤的实验研究  

Vascular endothelial growth factor shRNA mediated by pEGFP-H1 vector plasmid effectively inhibits glioma proliferation: an experimental study

作　　者：姜晓兵[1] 赵洪洋[1] 周凤[2] 刘如恩[3] 张方成[1] 赵甲山[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院神经外科,武汉430022 [2]深圳市眼科医院一病区 [3]中日友好医院神经外科

出　　处：《中华医学杂志》2005年第8期547-550,共4页National Medical Journal of China

摘　　要：目的　构建针对血管内皮生长因子(VEGF)的短发夹环RNA(shRNA)质粒(pEGFPH1 /VEGF),观察其在体内和体外对胶质瘤细胞株U251生长的抑制作用。方法　应用PCR构建携带绿色荧光蛋白(EGFP)基因的RNA干扰真核表达载体pEGFP- H1,并利用该载体介导VEGFshRNA。用阳离子脂质体转染U251细胞,通过荧光显微镜和流式细胞仪观察EGFP的表达,用RT- PCR检测VEGFmRNA的表达,以酶联免疫吸附实验(ELISA)检测培养液中VEGF蛋白的含量。同时筛选转染pEGFP- H1和pEGFP- H1 /VEGF的U251稳定细胞株,制备裸鼠U251细胞移植瘤模型,观察肿瘤生长情况。结果　与空载体转染组相比, pEGFP- H1 /VEGF对VEGF的抑制率达77. 9%。对照组和pEGFP -H1组裸鼠肿瘤重量分别为1 .7g±0 4g和1 .5g±0 .7g,体积分别为1573mm3 ±330mm3 和1430mm3 ±382mm3,两组之间重量和体积差异均无统计学意义(P>0 .05 ),而pEGFP- H1 /VEGF组肿瘤重量为0 .5g±0 .4g,体积为401mm3 ±272mm3,与对照组比较差异有统计学意义(P<0. 01)。结论　pEGFP H1介导的VEGFshRNA能有效抑制U251细胞中VEGF的表达,并在体内抑制肿瘤生长。Objective To investigate the inhibitory effects of RNA silencing via plasmid-mediated vascular endothelial growth factor (VEGF) shRNA on proliferation of glioma cells in vitro and in vivo. Methods pEGFP-H1/VEGF vector plasmid containing enhanced green fluorescent protein (EGFP) gene and expressing VEGF shRNA was constructed. The EGFP expression was detected by fluorescent microscopy and flow cytometry. Glioma cells of the line U251 were cultured and divided into 3 groups to be transfected with blank vector, pEGFP/H1plasmid, and pEGFP-H1/VEGF respectively. RT-PCR was used to detect the mRNA expression of VEGF in the supernatants of the culture media. The protein expression of VEGF in the supernatant was detected by ELISA. The cell growth was observed with MTT method. Fifteen nude rats were randomly divided into 3 equal groups to be transplanted with U251 cells and pEGFP-H1, U251 cells and pEGFP-H1/VEGF, and U251 cells only as blank control group. The growth of glioma was observed every day. 30 days after the rats were killed and the tumors were taken out to be examined. Results Twenty-four hours after transplantation, fluorescence microscopy showed great numbers of U251 cells that expressed green fluorescence in both the pEGFP-H1 group and pEGFP-H1/VEGF group. Flow cytometry showed that the rates of green fluorescent protein positive cells were 68.37% and 65.29% in these 2 groups (P>0.05). MTT method showed no significant difference in the effect on cell growth among the 3 groups (all P>0.05). PCR showed that the VEGF mRNA expression was significantly inhibited in the pEGFP-H1/VEGF group in comparison with those in the blank control group and the group transfected with pEGFP-H1 (both P<0.05). The concentration of VEGF protein of the U251 cells transfected with pEGFP-H1/VEGF was 530 ng/L.±118 ng/L, significantly lower than those of the control group (2571 ng/L±572 ng/L) and the group transfected with pEGFP-H1 (2402 ng/L±310 ng/L, by 77.9%) (both P<0.01). Tumor could be touched 13 days after transplantation

关 键 词：VEGF EGFP 血管内皮生长因子 肿瘤 介导 治疗 真核表达载体 U251细胞 RNA干扰 短发夹环RNA 

分 类 号：R73-36[医药卫生—肿瘤]