反义VEGF165基因转染对人神经母细胞瘤生物学行为的影响

目的探讨反义VEGF165基因转染在抑制肿瘤血管生成和肿瘤生长中的作用。方法构建正义和反义VEGF165真核表达载体，用脂质体转染人神经母细胞瘤细胞SH-SY5Y，G418筛选稳定表达细胞克隆。应用RT-PCR证实外源性反义VEGF mRNA的表达，免疫细胞化学和EUSA检测VEGF蛋白的表达水平，MTT法检测肿瘤细胞体外生长情况，并接种于裸鼠皮下，观察体内肿瘤增殖能力。结果RT-PCR证实转染反义VEGF的细胞中有外源性反义VEGF mRNA的表达，免疫细胞化学和EUSA显示VEGF蛋白表达明显降低，MTT实验显示转染前后细胞体外生长速度基本一致。而转染反义VEGF的肿瘤细胞裸鼠体内生长速度明显减慢。结论反义VEGF基因转染能够有效抑制SH-SY5Y细胞内源性VEGF蛋白的表达，抑制裸鼠体内肿瘤的生长。

EFFECT OF ANTISENSE VEGF165 cDNA TRANSFECTION ON THE GROWTH OF HUMAN NEUROBLASTOMA SH-SY5Y CELLS IN VITRO AND IN NUDE MICE

Objective The present study was designed to explore the inhibitory effect of antisense VEGF165 cDNA on angiogenesis, growth rate of human neuroblastoma. Methods The eukaryofic expression vectors bearing anfisense VEGF165 cDNA or sense VEGF165 cDNA were constructed. The stable cell lines transfected with the sense or anfisense VEGF165 cDNA were established by using the selective medium containing 400 mg/L of G418. These cell lines were further studied for the exogenous antisense VEGF mRNA expression by RT-PCR and inhibition of expression of endogenous VEGF protein by immunocytochemical staining and ELISA. The proliferation of the transfected cells was analyzed by MTr method. Transfected cells were subcutaneously transplanted into nude mice and the growth of tumor masses was observed and weighted. Results The antisense VEGF was only detected in SH-SY5Y/As VEGF cells by RT-PCR. The expression of VEGF protein was dramatically declined in the SH-SY5Y/ AsVEGF cells compared with the original cells and empty vector transfected cells by immunocytochemical staining and ELISA.There was no effect of antisense VEGF transfection on cell proliferation in vitro .The growth of tumor mass with the cells transfected with antisense VEGF was significantly decreased in nude mice. Conclusion Antisense VEGF cDNA transfection to SH- SYSY cells can effectively inhibit the expression of endogenous VEGF protein and tumor growth in nude mice.