靶向抑制皮层肌动蛋白装配对大肠癌细胞侵袭作用的影响

目的 构建能够靶向抑制细胞皮层蛋白装配的重组质粒,观察其对于肿瘤细胞侵袭作用的影响.方法 构建重组质粒EGFP/N2/Repeat,将其和空载体质粒转染入人大肠癌细胞Lo-Vo和HCT-8,分别扩增培养,绘制生长曲线,通过boyden小室模型观察大肠癌细胞侵袭性的改变.结果 实验获得与预期一致的DNA特异性片段,经双酶切和测序证实目的 片段正确的与载体质粒连接,重组质粒经过双酶切证实构建成功.显微镜下观察重组质粒成功转染人大肠癌细胞,细胞生长曲线提示转染与未转染该基因的细胞生长曲线相同;行细胞侵袭力实验,含有EGFP/N2/Repeat的癌细胞、空质粒转染细胞和对照组细胞(未转染)穿膜数分别为LoVo组:35.27±6.70、85.47±5.50、87.47±6.39;HCT-8组:15.07±2.34、26.73±4.43、26.07±3.67,含有拮抗分子的细胞穿膜数较其余两组明显减少,差异有统计学意义(P<0.01).结论 通过抑制皮层肌动蛋白装配可抑制大肠癌细胞的侵袭性.

Effect of target inhibiting the cortex protein assembly on invasive ability of colorectal cancer cells

Objective To construct a recombinant eukaryotic plasmid EGFP/N2/Repeat contai-ning the gene region-Repeat to study the impact of the gene on the invasion of the colon cancer cells. Methods Gene-Repeat ( a part of cortactin) was amplified using polymerase chain reaction ( PCR), then inserted into the fluorescence expression vector-EGFP/N2, after digestion and ligation using restriction en-donucleases and ligase. The correct expression vector EGFP/N2/Repeat and the empty vector EGFP/N2 were thansfected into LoVo and HCT-8 with liposome respectively. The metastasis of colon cancer cells was studied by using cell growth curve and transwell chambers culture respectively. Results The correct "Re-peat" clone was obtained and it was confirmed that Repeat was inserted into the expression vector correctly by using digestion identification and sequencing. Cell growth curves indicated that the cells LoVo/EGFP/ N2/Repeat (or HCT-8/EGFP/N2/Repeat) had the same curve as that of the cells LoVo (or HCT-8) and LoVo/EGFP/N2 (or HCT-8/EGFP/N2). Transwell chambers culture indicated the number of traversed membrane cells of LoVo/EGFP/N2/Repeat was less than that of the cells LoVo/EGFP/N2 and LoVo (35.27 ± 6.70 vs 85.47 ± 5.50 and 87.47 ± 6.39, P < 0.01 ), and that of HCT-8/EGFP/N2/Repeat, HCT-8/EGFP/N2 and HCT-8 was 15.07 ± 2.34,26.73 ± 4.43 and 26.07 ± 3.67, respectively ( P < 0.01 ). Conclusion The recombinant plasmid can inhibit the invasiveness of colon cancer cells through inhibiting cortical actin assembly.