沉默ILK基因的稳定转染细胞株的构建与鉴定

发布时间：2018-06-04 10:01

本文选题：ILK + RNA干扰　；参考：《现代口腔医学杂志》2015年01期

【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。
[Abstract]:Objective to study the function of integrin linked kinase (ILK) in cells, construct ILK gene sh RNA lentivirus vector, and identify its silencing effect in tongue squamous cell carcinoma cell line Tca-8113. Methods three pairs of ILK DNA pairs were designed and synthesized for three sites of the effective target sequence of ILK gene. After annealing, shILK-LV lentivirus vector was generated by ligating with linearized p ENTR/U6 vector to produce sh ILK-LV lentivirus vector. The positive clones were sequenced and identified by sh ILK-LV vector. The packaging plasmid Packaging Mix was co-transfected into 293T packaging cells to produce lentivirus. The virus titer was determined by the expression number of green fluorescent protein (GFP) in 293T cells and the appropriate MOI value was determined. Human tongue squamous cell carcinoma cell line Tca-8113 was infected with recombinant lentivirus. The expression of ILK in Tca-8113 cells was detected by Q PCR and Western blot. Results three ILK-sh RNA lentivirus vectors were successfully constructed by sequencing. After transfection into Tca-8113 cells, the expression of ILK in sh RNA-ILK-370 group was detected by sybr method, and 螖 Ct value was 0.268. Sh RNA-ILK-370 lentivirus vector was used to package the virus, the titer of the virus was determined, and when MOI=30-60 was used, the positive rate of the cells was the highest. The stable transgenic strain was screened with antibiotics after infection with virus, and the interference efficiency of Q PCR was 90.5%. The expression of ILK was obviously inhibited by Western blot. Conclusion the sh ILK-LV lentivirus vector was successfully constructed and a stable Tca-8113-sh ILK cell model was established, which provides a solid foundation for the study of the role of ILK in the signal transduction pathway of tongue squamous cell carcinoma.
【作者单位】：兰州大学第一临床医学院;兰州大学第一医院肿瘤外科;甘肃省肿瘤医院;兰州大学第一医院口腔科;
【基金】：甘肃省卫生厅卫生行业科研计划资助项目(GSWST2010-04)
【分类号】：R739.8

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