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论文摘要

shRNA慢病毒对人口腔鳞状细胞癌细胞端粒酶卡侯体蛋白1基因的沉默效应

Silencing effects of telomerase Cajal body protein 1-shRNA on human oral squamous cells

作者:胡玲 孙崇奎 苟雅萍 李燕 肖丽英

Author:Hu Ling, Sun Chongkui, Gou Yaping, Li Yan, Xiao Liying

收稿日期:2013-01-02          年卷(期)页码:2013,40(6):720-723

期刊名称:国际口腔医学杂志

Journal Name:International Journal of Stomatology

关键字:端粒酶卡侯体蛋白1,RNA干扰,基因沉默,

Key words:telomerase Cajal body protein 1,RNA interference,gene silencing,

基金项目:

国家自然科学基金青年基金资助项目(30901689);国家自然科学基金资助项目(81172579)

中文摘要

目的 探讨shRNA慢病毒对人口腔鳞状细胞癌Cal27细胞系端粒酶新蛋白,即端粒酶卡侯体蛋白1(TCAB1)基因的沉默效应,以期探索肿瘤基因治疗的新途径。方法 根据TCAB1基因,构建慢病毒shTCAB1-A~D,测定病毒滴度;用重组慢病毒体外感染Cal27细胞,荧光显微镜观察绿色荧光蛋白表达推断感染效率;半定量逆转录聚合酶链反应(RT-PCR)检测TCAB1 mRNA水平;Western blot检测TCAB1蛋白质的表达水平;四甲基偶氮唑盐比色(MTT)法检测细胞体外增殖能力;Transwell小室法检测细胞体外侵袭能力改变。结果 经PCR与测序鉴定证实成功构建靶向TCAB1的慢病毒RNAi载体,病毒滴度为1.5×108~3×108 U•mL-1。荧光显微镜观察显示,绝大部分细胞表达绿色荧光。与空白细胞对照组相比,4种不同的shTCAB1-A~D慢病毒感染组TCAB1 mRNA表达下调48.7%~62.0%;蛋白抑制率达45.6%~77.5%,shTCAB1-C组最明显。shTCAB1-C慢病毒能明显抑制Cal27细胞增殖能力,也能降低其侵袭能力,穿过人工基底膜的平均细胞数明显低于空白对照组及非特异性对照组,侵袭抑制率高达67.5%(P

英文摘要

Objective To investigate the inhibitory effects of telomerase Cajal body protein 1(TCAB1)-shRNA on oral squamous cell carcinoma Cal27 and to explore a novel cancer gene therapy approach by depletion of TCAB1 using RNA interference. Methods Four recombinant lentiviruses, called shTCAB1-A–D, which contain oligonucleotide(67 bp) targeting TCAB1 and green fluorescent protein EGFP Puro, were constructed based on different sites in the TCAB1 gene. A series of experiments were then carried out to demonstrate the silencing effects of the shTCAB1 lentiviruses on Cal27 cells. Semiquantitative analysis of TCAB1-mRNA expression levels was performed. TCAB1 protein expression level was analyzed using Western blot. Cell proliferation viability was tested by methye thiazolye telrazlium assay. Transwell invasion assay was used to evaluate cell invasiveness. Results Polymerase chain reaction and DNA sequencing demonstrated that the shTCAB1 lentiviral vectors were constructed successfully. The titers of the viruses ranged from 1.5×108 U•mL-1 to 3×108 U•mL-1. Compared with the blank control group, shTCAB1-A–D exhibited pronounced inhibitory effects, which significantly reduced both TCAB1 mRNA levels(48.7% to 62.0%) and protein levels(45.6% to 77.5%) in Cal27 cells. shTCAB1-C, the most effective inhibition group, caused slower growth of tumor cells and significant suppression of invasion potency(67.5%), (P

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