沉默ILK基因的稳定转染细胞株的构建与鉴定  

Construction and identification of stable transfected cell line for silencing ILK gene

作　　者：薛金才[1] 关泉林[2] 王千千[3] 王玉凤[1] 于涛[4] 丁方回 何等旗[4] 

机构地区：[1]兰州大学第一临床医学院,730000 [2]兰州大学第一医院肿瘤外科 [3]甘肃省肿瘤医院 [4]兰州大学第一医院口腔科

出　　处：《现代口腔医学杂志》2015年第1期7-11,共5页Journal of Modern Stomatology

基　　金：甘肃省卫生厅卫生行业科研计划资助项目(GSWST2010-04)

摘　　要：目的研究整合素连接激酶(ILK)在细胞中的功能,构建ILK基因sh RNA慢病毒载体,并对其在舌鳞癌细胞株Tca-8113中沉默效果进行鉴定。方法针对ILK基因有效靶序列的3个位点,设计合成3对oligo DNA,退火形成双链DNA,与线性化p ENTR/U6载体连接产生sh ILK-LV慢病毒载体,筛选出阳性克隆测序鉴定,用sh ILK-LV载体、包装质粒Packaging Mix共转染293T包装细胞,包装产生慢病毒,以293T细胞中绿色荧光蛋白(GFP)的表达数目测定病毒滴度并确定恰当的MOI值。获得重组慢病毒后感染人舌鳞癌细胞株Tca-8113,用q PCR及Western blot检测ILK在Tca-8113细胞中的表达。结果测序证实成功构建了3个ILK-sh RNA慢病毒载体,分别转染Tca-8113细胞后用sybr法检测出sh RNA-ILK-370组ILK的表达明显下降,ΔΔCt值为0.268。用sh RNA-ILK-370慢病毒载体包装病毒,测定病毒滴度,并得出当MOI=30-60时,细胞阳性比率最高。用病毒感染Tca-8113细胞后再用抗生素筛选稳转株,后用q PCR检测干扰效率达90.5%,Western blot检测ILK表达明显被抑制。结论成功构建sh ILK-LV慢病毒载体并建立了稳定的Tca-8113-sh ILK细胞模型,为研究ILK在舌鳞癌信号转导通路中的作用提供了坚实基础。Objective To study the function of integrin-linked kinase （ILK） in the cell, construct ILK gene shRNA lentiviral vector, and detect silencing effect of ILK in tongue squamous cell carcinoma cell line Tea-8113. Methods The target to three sequences of ILK gene, designed and synthesis three pairs of oligo DNA, anneal to double- stranded DNA, and connect the linearized vector pENTR/U6 to generate shlLK-LV lentiviral vectors , select positive clones and sequence. Using shlLK-LV vector and packaging plasmid packaging Mix cotransfection of 293T cells to produce lentivirus. The expressing of green fluorescent protein （GFP） in 293T cell was measured to virus titer to determine the appropriate MOI value. Infect human tongue squamous cell carcinoma cell line Tea-8113 after obtaining recombinant lentivirus. ILK expression in Tea-8113 cells by qPCR and Western blot analysis. Results The sequencing confirmed successfully constructed three ILK-shRNA lentiviral vectors, Tca-8113 cells were transfected by these respectively and shRNA-ILK-370 group ILK ＂s were detected by the method of sybr decreased apparently. AACt value of 0.268. With shRNA-ILK-370 lentiviral vector packaging virus, detect viral titer , and when MOI = 30-60 , the positive cells was highest. Infeting Tea-8113 cells were then screened by antibiotic strains, the interference efficiency was 90.5% using qPCR, the ILK expression was significantly inhibited by Western blot. Conclusion Successfully constructed shlLK-LV lentiviral vector and established a stable Tca-8113-shlLK cell model, providing a solid foundation for the study of the role of ILK in tongue squamous cell signal transduction pathway.

关 键 词：ILK RNA干扰 舌鳞癌 基因表达 

分 类 号：R739.8[医药卫生—肿瘤]