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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 construct the eukaryotic expression vector of human Twist gene and to establish a human cervical cancer cell line HCC94 stably expressing Twist. The biological behavior of HCC94 cells before and after transfection was studied. Methods: Twist open reading frame was amplified by RT-PCR from HCE1 cells. The PCR products were cloned into pZsGreen-C1 eukaryotic expression vector and constructed and identified as pZsGreen-C1 / Twist. The recombinant plasmid was transfected into human HCC94 cells with lipofectamine 2000. The stable and highly expressed Twist cell clone was screened by G418 and detected by fluorescence, Western blot and Realtime RT-PCR. MTT assay and invasion chamber Experimental study of HCC94 cells proliferation, adhesion and migration before and after transfection. Results: The eukaryotic expression vector pZsGreen-C1 / Twist was successfully constructed, and the p-tw-1 clone stably expressing human Twist was screened out. The transfection efficiency was> 70% by fluorescence microscopy. Realtime RT-PCR amplification curves and melting point curves showed that Twist gene was effectively amplified, and the relative expression levels of mRNA in p-tw-1 group, p-tw-v (transfected empty vector) group and HCC94 Were 4.14 ± 2.26, 2.03 ± 1.10 and 1.90 ± 0.78 respectively. The level of p-tw-1 was significantly up-regulated compared with the other two groups, the difference was statistically significant, F = 10.24, P = 0.001; Significance of learning, P = 0.146. The results of Western blot showed that the Twist gene was stably expressed, and the ratios of IOD to GAPDH in p-tw-1, p-tw-v and HCC94 groups were 0.65, 0.33 and 0.30, respectively. The growth of p-tw-1 cells from day 4 was faster than that of p-tw-v and HCC94 groups, the difference was statistically significant, F = 24.113, P = 0.000; however, there was no significant difference between the two groups Meaning, P = 0.229. The average numbers of transmembrane cells in P-tw-1, P-tw-v and HCC94 groups were (263.67 ± 18.04), (126.00 ± 17.69) and (116.33 ± 9.87) , The difference was statistically significant, F = 83.093, P = 0.000; There was no significant difference between p-tw-v and HCC94 group, P = 0.478. The average A values of the adherent cells in P-tw-1, P-tw-v and HCC94 groups were 0.38 ± 0.01, 0.53 ± 0.01 and 0.57 ± 0.01 respectively, while the A value in p-tw-1 group was significantly lower F = 65.086, P = 0.000; There was no significant difference between p-tw-v group and HCC94 group (P = 0.244). CONCLUSIONS: Twist gene overexpression enhances the proliferation and migration of cervical cancer cells and decreases its adhesion ability. Twist eukaryotic expression vector and stable expression of Twist HCC94 cell line lay the foundation for the further study of Twist function.