摘要
目的利用小干扰RNA(siRNA)技术沉默Nestin基因,观察其逆转胶质瘤细胞恶性表型的效果。方法设计靶向Nestin基因的siRNA片段,利用脂质体转染人U251胶质瘤细胞株,逆转录一聚合酶链反应(RT—PCR)和免疫组织化学法检测Nestin基因和蛋白的表达,噻唑蓝(MTF)比色法检测细胞增殖,Transwell实验检测细胞的迁移能力,软琼脂实验检测细胞的恶性增殖能力。结果转染siRNA基因片段的U251胶质瘤细胞株基因表达水平(U251细胞为0.83±0.16、U251/vect细胞为0.81±0.23、U251/S3为0.20±0.11)和蛋白表达水平(U251细胞为157.73±4.12、U251/vect细胞为153.34±5.27、U251/S3为112.20±3.16)显著下降(P〈0.05),MTT实验显示siRNA干扰后U251胶质瘤细胞株体外增殖速度减慢、细胞侵袭能力及软琼脂集落形成能力均显著降低(P〈0.01)。结论Nestin基因表达下调可抑制胶质瘤细胞株U251的侵袭及增殖能力。
Objective To investigate the therapeutic efficacy of siRNA fragments silencing Nes- tin,which may inhibits the growth and invasion of glioma cells. Methods The siRNA sequence fragments targeting Nestin were designed and transferred into human glioma cell line U251. Reverse transcription-poly- merase chain reaction ( RT-PCR ) and immunochemistry method were used to explore the expression of Nes- tin. The tumor-suppressing effect of silencing Nestin on human glioma cells was assessed by methyl thiazol tetrazolium (MTF) and transwell chamber assays. Soft agar clone formation was adopted to identify oncoge- nicity. Results Nestin mRNA expression levels in U251 cells,U251/vect cells,U251/S3 cells were 0. 83 ± 0. 16,0. 81 ± 0. 23,0. 20 ± 0. 11. Nestin protein expression levels in U251 cells, U251/vect cells, U251/$3 ceils were 157.73 ± 4. 12,153.34 ± 5.27,112. 20 ± 3.16. MTF assays showed that the cell growth rate of U251 was significantly inhibited after transferred siRNA sequence fragments. The cellular adhesion ability and oncogenicity were weakly relevant. Conclusion Downregulation expression of Nestin can inhibits the growth of the human glioma cell line U251.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2012年第9期1677-1679,共3页
Chinese Journal of Experimental Surgery