沉默α-catulin基因的人舌鳞状细胞癌TSCCA细胞模型的建立

doi:10.13481/j.1671-587x.20160312

摘要/Abstract

摘要：

目的: 设计并构建α-catulin-shRNA表达载体,建立沉默α-catulin基因的人舌鳞状细胞癌(TSCC),TSCCA细胞模型并观察其沉默效率,为TSCC的基因治疗提供依据。方法: TSCCA细胞分为转染组(转染sh-catu1、sh-catu2、sh-catu3和sh-catu4质粒)、阴性对照组(转染sh-nc质粒)及空白对照组(未处理)。根据GenBank上获取的α-catulinmRNA序列,设计并合成4条α-catulin-shRNA质粒表达载体。测序鉴定后,经脂质体介导,将3组质粒分别转染至体外培养的人TSCCA细胞中,荧光显微镜观察各组TSCCA细胞中绿色荧光蛋白(GFP)的表达;采用RT-PCR和Western blotting法检测TSCCA细胞中α-catulin基因的mRNA及其蛋白相对表达水平及沉默效率。结果: 测序鉴定表明成功构建了编码α-catulin的shRNA质粒表达载体sh-catu1、sh-catu2、sh-catu3和sh-catu4;转染TSCCA细胞后,荧光显微镜下观察到表达载体在细胞中成功表达。RT-PCR检测,与空白对照组比较,转染组细胞中α-catulinmRNA表达水平降低(P<0.05),沉默效率升高(P<0.05);与阴性对照组比较,转染组细胞中α-catulinmRNA相对表达水平降低(P<0.05),沉默效率升高(P<0.05)。Western blotting法检测,与空白对照组比较,转染组细胞中α-catulin蛋白相对表达水平降低(P<0.05),沉默效率升高(P<0.05);与阴性对照组比较,转染组细胞中α-catulin蛋白相对表达水平降低(P<0.05),沉默效率升高(P<0.05)。结论: 成功构建α-catulin-shRNA重组质粒载体,建立了沉默α-catulin基因的人TSCC的TSCCA细胞模型,为研究α-catulin基因在TSCC发病机制中的作用提供了细胞模型。

关键词: 癌, 鳞状细胞, 舌肿瘤, &alpha, -catulin基因, TSCCA细胞, 细胞转染

Abstract:

Objective: To design and construct the expression vectors of α-catulin-shRNA,and to establish the TSCCA cell model with silencing α-catulin gene of tongue squamous cell carcinoma (TSCC) and observe its silencing efficiency,and to provide foundation for gene treatment of TSCC. Methods: The TSCCA cells were divided into transfection groups (transfected with sh-catu1,sh-catu2,sh-catu3, and sh-catu4),negative control group (transfected with sh-nc),and blank control group(untreated).According to the mRNA sequence of α-catulin obtained from GenBank, the expressing vectors of α-catulin-shRNA plasmids were designed and compounded.After the vectors were identified by DNA sequencing,the expressing vectors were transfected into the TSCCA cells cultured in vitro after mediated with liposome.The expression of green fluorescence protein (GFP) in TSCCA cells was observed by fluorescence microscope;the relative expression levels and silencing efficiency of α-catulin mRNA and protein were detected by RT-PCR and Western blotting methods. Results: The DNA sequencing results showed the plasmid expression vectors containing shRNA against α-catulin gene (sh-catu1,sh-catu2,sh-catu3,and sh-catu4) were constructed correctly.After transfection,the expressing vectors were expressed successfully in the TSCCA cells.The RT-PCR results showed that compared with blank control group,the relative expression levels of α-catulin mRNA in TSCCA cells in ttansfection groups were decreased (P<0.05) and the silencing efficiencies were increased (P<0.05).Compared with negative control group,the relative expression levels of α-catulin mRNA in TSCCA cells in transfection groups were decreased (P<0.05) and the silencing efficiencies were increased (P<0.05).The Western blotting results showed that compared with blank control group,the relative expression levels of α-catulin protein in TSCCA cells in transfection groups were decreased (P<0.05) and the silencing efficiencies were increased (P<0.05).Compared with negative control group,the relative expression levels of α-catulin protein in TSCCA cells in transfection groups were decreased (P<0.05) and the silencing efficiencies were increased (P<0.05). Conclusion: The recombinant plasmid vectors are constructed successfully,which could silence the α-catulin expression in TSCCA cells.This study will provide a new cell model for further study of the role of α-catulin in the pathogenesis of TSCC.

Key words: carcinoma,squamous cell, tongue neoplasms, α-catulin gene, TSCCA cell, cell transfection

中图分类号:

R739.86

韩春耀, 张斌, 马雪, 刘明媛, 薛中原, 郝丽静, 葛树卿. 沉默α-catulin基因的人舌鳞状细胞癌TSCCA细胞模型的建立[J]. 吉林大学学报(医学版), 2016, 42(03): 481-485.

HAN Chunyao, ZHANG Bin, MA Xue, LIU Mingyuan, XUE Zhongyuan, HAO Lijing, GE Shuqing. Establishment of tongue squamous cell carcinoma TSCCA cell model with silencing α-catulin gene[J]. Journal of Jilin University Medicine Edition, 2016, 42(03): 481-485.

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