兰州大学机构库 >学院待认领
沉默ILK基因的稳定转染细胞株的构建与鉴定
Alternative TitleConstruction and identification of stable transfected cell line for silencing ILK gene
薛金才
Thesis Advisor关泉林
2014-05-26
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name硕士
KeywordILK RNA干扰 舌鳞癌 基因表达
Abstract目的:研究整合素连接激酶(ILK)在细胞中的功效,构建ILK基因shRNA慢病毒载体,并对其在舌鳞癌细胞株Tca-8113中沉默效果进行判定。方法:针对ILK基因有用靶序列的3个位点,计划合成3对oligo DNA,退火构成双链DNA,与线性化pENTR/U6载体衔接发生shILK-LV慢病毒载体,筛选出阳性克隆测序判定,用shILK-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qPCR及Western blot检测ILK在Tca-8113细胞中的表达。结果:测序证明成功构建了3个ILK-shRNA慢病毒载体,分别转染Tca-8113细胞后用sybr法检测出shRNA-ILK-370组ILK的表达明显下降,ΔΔCt值为0.268。用shRNA-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qPCR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shILK-LV慢病毒载体并建立了稳定的Tca-8113-shILK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。
Other AbstractObjective: 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 Tca-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 shILK-LV lentiviral vectors , select positive clones and sequence. Using shILK-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 Tca-8113 after obtaining recombinant lentivirus. ILK expression in Tca-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. ΔΔCt 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 Tca-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. Conclusions: successfully constructed shILK-LV lentiviral vector and established a stable Tca-8113-shILK cell model, providing a solid foundation for the study of the role of ILK in tongue squamous cell signal transduction pathway.
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Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/223740
Collection学院待认领
Affiliation临床医学院
Recommended Citation
GB/T 7714
薛金才. 沉默ILK基因的稳定转染细胞株的构建与鉴定[D]. 兰州. 兰州大学,2014.
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