腺病毒载体介导的Polo样激酶1短发夹环RNA对胶质瘤体内外增殖的抑制作用

目的探讨重组腺病毒载体介导的Polo样激酶1（PLK1）短发夹环RNA（shRNA）对人胶质瘤细胞株U251在体内外增殖的抑制作用。方法设计并合成针对PLK1的shRNA，并克隆至真核表达载体pEGFP—H1上，再将H1启动子和shRNA亚克隆至腺病毒穿梭质粒pAdTrack上，通过在携带有pAdeasy-1质粒的BJ5183细菌内同源重组，生成针对PLK1的shRNA重组腺病毒载体pAD-H1／PLK1，经293细胞包装产生重组腺病毒AD-H1／PLK1，感染U251细胞。应用逆转录-聚合酶链反应（RT—PCR）的方法检测U251细胞PLK1mRNA表达，流式细胞仪检测细胞凋亡以及细胞G2／M期转变情况，以噻唑蓝（MTT）比色法检测各组细胞的增殖活性。同时制备裸鼠U251细胞移植瘤模型并注射重组腺病毒，观察肿瘤生长情况。结果成功构建针对PLK1基因的RNA干扰腺病毒表达载体，与AD-H1组比较AD-H1／PLK1组在转染24h和48hU251细胞PLK1mRNA表达水平分别降低50．4％（P〈0．01）和71．9％（P〈0．01）。且感染AD-H1／PLK1病毒48h后的U251细胞凋亡率从8．3％升至65．6％（P〈0．01），而G2／M期的细胞从21．5％增至51．4％（P〈0．01），细胞的增殖能力则下降50％（P〈0．01）。体内实验表明重组腺病毒载体介导的PLK1shRNA也明显抑制肿瘤生长（P〈0．01）。结论腺病毒介导的RNA干扰技术可有效降低PLK1在胶质瘤细胞中的表达水平，抑制肿瘤细胞在体内外的增殖活性。

Inhibitory effects of adenovirus mediated polo-like kinase-1 (PLK1) shRNA on proliferation of U251 cells in vitro and in vivo

Objective To investigate the inhibitory effects of adenovirus mediated polo-like ki-nase-1 (PLK1) shRNA on proliferation of U251 cells in vitro and in vivo. Methods Short hairpin RNA (shRNA) targeting to PLK1 gene was designed and synthesized and cloned into eukaryotic expression vector pEGFP-H1,H1 promoter and shRNA were subcloned into adenoviral shuttle plasmid pAdTrack, and this plasmid was recombined with pAdeasy-1 in bacteria BJ5183 to produce plasmid pAdTrack-H1/ PLK1. Plasmid pAdTrack-H1/PLK1 was transfected into 293 cells to pack the adenoviruses, and AD-H1/PLK1.U251 cells were infected with this recombinant adenovirus. The expression of PLK1 mRNA in U251 cells was detected by using RT-PCR,apoptosis and cell cycle transition by FACS,and MTT assay was used to detect cells viability. The recombinant viruses were injected into the xenograft tumors of U251 cells in nude mice, then the tumor growth was observed. Results The recombinant adenoviral vector carrying PLK1 shRNA was constructed successfully. PLK1 mRNA expression in U251 cells was decreased remarkably by 50.4%(P<0.01) and by 71.9% respectively 24 h and 48 h after transfection with AD-H1/PLK1 (P< 0.01). Accordingly, AD-H1/PLK1 elevated the apoptosis rate of U251 cells from8.3% to 65.6% (P< 0.01), increased G2/M phase cells ratio from 21.5% to 51.4% (P< 0.01),and reduced cell proliferation by 50% 48 h after transfection respectively. Adenovirus mediated PLK1 shRNA also inhibited the growth of U251 cell in vivo. Conclusion Adenovirus mediated PLK1 shRNA can inhibit the expression of PLK1 in glioma cells effectively and suppress growth of U251 cell in vitro and in vivo.