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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PLK1 mRNA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24 h和48 h U251细胞PLK1 mRNA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48 h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PLK1 shRNA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。
Objective To investigate the inhibitory effect of recombinant adenovirus vector-mediated Polk-like kinase 1 (PLK1) short hairpin RNA (shRNA) on proliferation of human glioma cell line U251 in vitro and in vivo. Methods shRNA targeting PLK1 was designed and synthesized and cloned into the eukaryotic expression vector pEGFP-H1. The H1 promoter and shRNA were subcloned into the adenovirus shuttle plasmid pAdTrack, and then transfected into pAddeasy-1 plasmids in BJ5183 bacteria Homologous recombination, shRNA recombinant adenoviral vector pAD-H1 / PLK1 for PLK1 was generated, and the recombinant adenovirus AD-H1 / PLK1 was packaged by 293 cells to infect U251 cells. The expression of PLK1 mRNA in U251 cells was detected by reverse transcriptase-polymerase chain reaction (RT-PCR), the apoptosis and G2 / M phase transition in U251 cells were detected by flow cytometry and MTT assay Group cell proliferation activity. At the same time, nude mouse U251 cell xenograft models were prepared and injected with recombinant adenovirus to observe tumor growth. Results Compared with AD-H1 group, the expression of PLK1 mRNA in U251 cells decreased by 50.4% (P <0. 05) at 24 h and 48 h after AD-H1 / PLK1 transfection. .01) and 71.9% (P <0.01). Moreover, the apoptosis rate of U251 cells infected with AD-H1 / PLK1 virus increased from 8.3% to 65.6% (P <0.01) at 48 h, while the cells in G2 / M phase increased from 21.5% to 51.4% (P <0.01), while the cell proliferation decreased by 50% (P <0.01). In vivo experiments showed that recombinant adenovirus vector-mediated PLK1 shRNA also significantly inhibited tumor growth (P <0.01). Conclusion Adenovirus-mediated RNA interference can effectively reduce the expression of PLK1 in glioma cells and inhibit the proliferation of tumor cells in vitro and in vivo.