针对核干细胞因子腺病毒干扰载体构建与干扰作用观察  

Construction of RNAi adenovirus targeting nucleostemin and its gene silencing function

作　　者：韩崇旭 孙艳[2] 许文荣[3] 张锡然[4] 

机构地区：[1]江苏省苏北人民医院·扬州大学临床医学院临床医学检测中心,江苏扬州225001 [2]江苏省苏北人民医院·扬州大学生殖医学中心,江苏扬州225001 [3]江苏大学基础医学与医学技术学院检验医学研究所,江苏镇江212013 [4]南京师范大学生命科学院分子细胞生物学研究所,江苏南京210023

出　　处：《中华肿瘤防治杂志》2015年第8期588-594,共7页Chinese Journal of Cancer Prevention and Treatment

基　　金：江苏省自然科学基金(BK2007072);江苏省卫生厅“科教兴卫工程”医学重点学科“实验诊断学”开放性课题(XK200723/WKF0809);江苏省苏北人民医院院内基金(yzucms201039)

摘　　要：目的构建针对核干细胞因子(Nucleostemin)基因寂寞的RNA干扰腺病毒载体,观察其干扰作用。方法根据克隆的全长Nucleostemin基因(1 650bp,GenBank接受序号:AY825265)设计RNA干扰反向重复序列,采用分子生物学组装腺病毒载体,制备高效价RNAi重组腺病毒。实验分为Backbone-P1P2重组腺病毒感染组(P1P2干扰组)、Backbone-P3P4重组腺病毒感染组(P3P4干扰组)、Backbone空质粒病毒感染对照组(B组)和无病毒感染对照组。不同剂量腺病毒液RNAi(分别为60、30、15和7.5μL)转染F6细胞及U937细胞,采用蛋白质印迹法检测F6细胞及U937细胞中核干细胞因子的蛋白表达水平,采用实时荧光定量PCR(QPCR)检测U937细胞中核干细胞因子的mRNA表达水平。计数法绘制细胞生长曲线,判定细胞活力;流式细胞术检测细胞周期变化。结果靶向核干细胞因子基因的腺病毒RNA干扰载体(Backbone-P1P2,Backbone-P3P4)被成功构建。蛋白质印迹法检测结果显示,F6细胞内不同梯度浓度腺病毒RNA干扰后,核干细胞因子表达量分别为1.23、4.48、5.03和5.75,随P3P4片段RNA干扰浓度梯度下降而核干细胞因子表达量呈上升趋势。P1P2干扰组蛋白水平为0.219±0.051,与空质粒转染对照组的2.174±0.196比较明显降低,F=279.4,P<0.001;P3P4干扰组蛋白水平为0.0164±0.003,与空质粒转染对照组比较明显降低,F=363.4,P<0.001。P1P2干扰组基因表达水平为1.585±0.20,与空质粒转染对照组的3.932±0.271比较明显降低,F=145.9,P<0.001;P3P4干扰组基因表达水平为1.328±0.251,与空质粒转染对照组比较明显降低,F=149.9,P<0.001。细胞周期实验发现,核干细胞因子干扰后,P1P2腺病毒RNA干扰组为44.89±4.04,与空质粒转染对照组的54.74±3.28对比减低,F=10.75,P=0.031;P3P4干扰组为45.00±3.82,与空质粒转染对照组对比减低,F=11.23,P=0.029;S期+G2M期细胞P1P2干扰组为55.11±2.81,与空质粒转染对照组的45.26±1.72对比升高,F=26.82,P=0.007;P3P4OBJECTIVE To construct recombinant adenovirus vector carrying a small interfering RNA targeting nu- cleostemin gene and to elucidate its inhibiton effect on tumor cells. METHODS The RNA interference inverted repeat se- quence was designed according to the full-length nucleostemin gene of self-reported in GenBank （1 650 bp, GenBank accepted number： AY825265）,construction of recombinant adenovirus vector using molecular biology methods. Recombi- nant adenovirus vector was obtained by co-transfect AD293 cells and the high titer virus liquid was acquired by repeated freeze-thaw AD293 cell lysis. Experiment was divided into four groups namely Backbone-PIP2 adenovirus infection group, Backbone-P3P4 adenovirus infection group, Empty plasmid viral infection group and non-infected group. Cell prolif- erations were observed by transfected different doses（60,30,15 and 7.5 μL） of adenovirus RNAi into F6 cells and U937 cells. The protein expression and the mRNA levels of nucleostemin in F6 cells and U937 cells were detected by Western Blotting and real-time PCR respectively. The cell growth curve was drawn by cell counting method,and the change of cell cycle was observed by Flow Cytometry. RESULTS Adenoviral vector （Backbone-PlP2,Backbone-P3P4） was constructed successfully for target of nucleostemin by RNAi. Nucleostemin protein showed a gradient decreased when different doses of adenovirus RNAi transfected into F6 cells, and the expressions of nucleostemin were 1.23,4.48,5.03,5.75, respective ly. Protein levels of P1P2 in inhibition group were 0. 219±0. 051,P3P4 in inhibtion group was 0. 0164±0. 003 and in control group of empty plasmid transfection group was 2. 174±0. 196. Backbone-PiP2 adenovirus infection group was sig- nificantly lower than that in empty plasmid viral infection group （F= 279.4 ,P〈0. 001） ,Backbone-P3P4 in adenovirus in- fection group was significantly lower than that in empty plasmid viral infection group （F= 363.4, P〈0. 001）. The gene expression of P1P2 in inhibition grou

关 键 词：核干细胞因子 RNA干扰 腺病毒 表达载体 基因表达 细胞周期 细胞增殖 

分 类 号：R73-3[医药卫生—肿瘤]