摘要
目的:构建人Twist基因真核表达载体,并建立稳定高表达Twist的人宫颈癌HCC94细胞系,研究转染前后HCC94细胞生物学行为。方法:用RT-PCR法扩增人低分化宫颈癌HCE1细胞中Twist开放阅读框架,将PCR产物克隆到pZsGreen-C1真核表达载体,构建重组质粒pZsGreen-C1/Twist并鉴定。采用脂质体法把重组载体转染入人高分化宫颈癌HCC94细胞,经G418筛选获得稳定高表达Twist的细胞克隆,并进行荧光,蛋白质印迹法和Realtime RT-PCR检测;MTT法和侵袭小室实验研究转染前后HCC94细胞的增殖、黏附和迁移能力。结果:成功构建pZsGreen-C1/Twist真核表达载体,筛选出稳定高表达人Twist的p-tw-1克隆,经荧光显微镜观察其转染效率>70%。Realtime RT-PCR扩增曲线和熔点曲线显示,Twist基因有效扩增,p-tw-1组、p-tw-v(转染空载体)组和HCC94(未转染)组mRNA相对表达量分别为4.14±2.26、2.03±1.10和1.90±0.78,p-tw-1组相对于其他两组水平明显上调,差异有统计学意义,F=10.24,P=0.001;而对照组之间差异无统计学意义,P=0.146。蛋白质印迹法检测结果显示,Twist基因稳定表达,p-tw-1组、p-tw-v组和HCC94组IOD与GAPDH比值分别为0.65、0.33和0.30。p-tw-1组细胞从第4天起生长趋势快于p-tw-v组和HCC94组,差异有统计学意义,F=24.113,P=0.000;而两对照组之间差异无统计学意义,P=0.229。P-tw-1、P-tw-v和HCC94组平均穿膜细胞数分别为(263.67±18.04)、(126.00±17.69)和(116.33±9.87)个,p-tw-1组细胞数明显增多,差异有统计学意义,F=83.093,P=0.000;p-tw-v与HCC94组之间差异无统计学意义,P=0.478。P-tw-1、P-tw-v和HCC94组黏附细胞平均A值分别为0.38±0.01、0.53±0.01和0.57±0.01,p-tw-1组A值明显降低,差异有统计学意义,F=65.086,P=0.000;p-tw-v组与HCC94组之间差异无统计学意义,P=0.244。结论:Twist基因高表达可增强宫颈癌细胞增殖和迁移能力,降低其黏附能力;Twist真核表达载体和稳定高表达Twist的HCC94细胞系构建为进一步研究Twist功能奠定了基础。
OBJECTIVE：To analyze the biological behavior of HCC94 cells after transfection,build the human Twist gene eukaryotie expression vector and establish a stable high expression of Twist in human cervical carcinoma HCC94 cell lines. METHODS：Open reading frame of Twist gene was amplificated by RT-PCR from human poorly differentiated cervi- cal cancer cells HCE1 and inserted into pZsGreen-C1 to construct the eukaryotic expression plasmid pZsGreen-C1/Twist. Using lipofectaminWMS000,the plasmid pZsGreen-C1/Twist was transfected into human well-differentiated cervical cancer celIs HCC94. The stable cell lines that expressed the truncated Twist isoforms was screened by G418 and identified with fluorescence,western blot and realtime RT-PCR. The ability of cell proliferation,adhesion and migration were analyzed by MTT method and transwell chamber assay to compare with untransfected cell. RESULTS： The eukaryotie expression plas- mid pZsGreen-C1/Twist was successfully constructed and the stable HCC94 cell lines p-tw-1 was obtained that expressed open reading frame of Twist gene. The efficiency of transfection more than 70 percent was observed by fluorescence micro- scope. Effective amplification of Twist gene was showed by amplification plot and melt curve of realtime RT-PCR.Expression of mRNA of p-tw-1, p tw-v（transfected with empty vector） and HCC94 （transfect nothing） was 4.14±2.26, 2.03 ± 1.10,1.90 ± 0.78, the level of p tw-1 raised significantly, the difference was statistically significant （F= 10.24, P = 0. 001） while no significant difference between the two control groups （P〈 0. 146）. Stable expression of Twist gene showed by western blot,the ratio of IOD and GADPH was 0.65,0.33 and 0.30 respectively. The growth trend of p tw-1 was faster than the two control groups p-tw-v and HCC94 from the fourth day,the difference was statistically significant （F= 24.113 ,P= 0. 000） while no significant difference between the two control groups （P=0. 229） ;the average number of transmembrane cell including p-tw 1,p-tw-v,HCC94 was 263.67± 18.04,126.00±17.69,116. 33±9.87,the number of cell increased significantly in p-tw-1, the difference was statistically significant （F= 83. 093, P= 0. 000） while no signif- icant difference between the two control groups （P=0. 478） ;the average A value of adherent cells including p-tw 1 ,p-tw- v,HCC94 was 0.38±0.01,0.53±0.01,0.57i0.01,and A value of group p tw-1 was significantly lower. The difference was statistically significant （F=65. 086,P=0. 000） while no significant difference between the two control groups （P= 0. 244）. CONCLUSIONS： The overexpression of Twist can enhance the capability of proliferation and migration of cervical cancer cells while reduce its ability of adhesion. The eukaryotic expression plasmid pZsGreen-C1/Twist and stable expression of Twist HCC94 cell lines established the foundation of further research for the function of Twist gene.
出处
《中华肿瘤防治杂志》
CAS
北大核心
2013年第3期185-190,共6页
Chinese Journal of Cancer Prevention and Treatment
基金
海南省教育厅高校科学研究资助项目(Hjkj2011-33)
