Polo-like kinase 1短发卡RNA重组腺病毒载体构建及其生物学应用  

Construction of the recombinant adenovirus mediated short hairpin RNA to silence Polo - like kinase 1 and its biomedical application

作　　者：李泉[1] 毛永欢 余翔[1] 蔡则灵 王伟林[1] 喻春钊[1] 

机构地区：[1]南京医科大学第二临床医学院普外科,210029

出　　处：《中华实验外科杂志》2016年第1期54-57,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金（30972910、81172269）;中国博士后一等基金（20060390294）;江苏省自然科学基金（BK2011858）;江苏省六大人才高峰基金（WSW-032）

摘　　要：目的构建针对人类Polo-like kinase1（Plk1）基因的短发卡RNA（shRNA）真核表达载体，并进行腺病毒包被。方法设计并合成含有小发夹结构的Plk1 shRNA对应模板序列，退火并与pYr-1．1载体重组质粒；通过LR体外同源重组将pYr-1．1-Plk1—shRNA表达框构建至pAd—DEST腺病毒表达载体上；包装重组腺病毒；感染人类胰腺癌细胞BxPC-3验证干扰效率。实验分3组：对照组（Control）、空载组[rAd-增强绿色荧光蛋白（tAd—EGFP）]、实验组（rAd—shPlk1），实时定量反转录聚合酶链反应（RT-qPCR）和Western blot检测Plk1基因的表达，流式细胞术检测细胞周期的变化及对细胞凋亡的影响。结果腺病毒感染BxPC-3细胞后，RT-qPCR检测Plk1 mRNA表达量：对照组1．047±0．315、空载组1．121±0．199、实验组0．119±0．050；Western blot检测Plk1蛋白表达量：对照组0．760±0．088、空载组0．743±0．101、实验组0．050±0．043，实验组较对照组及空载组Plk1 mRNA和蛋白水平均明显降低（P〈0．01）。流式细胞术检测G2期细胞比例：对照组6．077±0．056、空载组6．533±1．644、实验组33．427±7．774，实验组G：期细胞数比例显著高于空载组和对照组（P〈0．01）。感染24h后实验组的细胞凋亡率明显高于空载组和对照组（P〈0．01）。结论成功构建包被有Plk1 shRNA的腺病毒表达载体，且重组腺病毒载体可以抑制Plk1基因的表达，引起细胞周期G2／M期停滞和促进细胞凋亡。Objective To construct the recombinant adenovirns mediated short hairpin RNA （shRNA） to silence Polo - like kinase 1. Methods Four pairs of shRNA targeting different regions of the Plk1 transcript were synthesized with the vector pYr - 1.1 （hU6/EGFP/Neo）. After gene sequence analysis, pYr - 1.1 - Plk1 - shRNA2 and pYr - 1.1 - Plk1 - shRNA4 were combined to adenovirus vector pAd/ PL- DEST through homologous recombination in vitro. After package and screen, pancreatic cancer cell BxPC - 3 was infected by these recombinant adenoviruses to verify their interference efficiency. Real - time quantitative reverse transcriptase - polymerase chain reaction （ RT - qPCR） was used to detect Plk1 mRNA level, and Western blotting was used to detect Plk1 protein level. Cell cycle and apoptosis were determined by flow eytometry. Results After infection, the levels of Plk1 mRNA （control, 1.047 ± 0.315; rAd-EGFP, 1. 121 ±0. 199; rAd - shPlk1, 0.119 ± 0.050） and protein （control, 0.760 ± 0.088; tad - EGFP, 0. 743 ± 0. 101 ; tad - shPlk1, 0. 050 ± 0. 043 ）. The expression levels of Plk1 mRNA and protein in rAd - shplkl group were significantly lower than in the control and rAd - EGFP groups. Cells in tad - shPlk1 groupd were arrested in G2/M phase （ P 〈 0. 01 ） and apoptosis rate in tad - shPlk1 group was significantly higher than in other two groups （ P 〈 0.01 ）. Conclusion The recombinant adenovirns mediated shRNA to silence Polo - like kinasel is successfully constructed, and inhibition of Plk1 can arrest cells in G2/M phase and induce apoptosis.

关 键 词：Polo-like KINASE 1 短发卡RNA 重组腺病毒 胰腺癌 RNA干扰 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]