摘要
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人人大肠癌细胞LoVo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。
Objective To construct a recombinant eukaryotic plasmid EGFP/N2/Repeat containing 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 endonucleases 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.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2009年第2期156-158,共3页
Chinese Journal of Experimental Surgery
基金
基金项目：国家自然科学基金资助项目（30771126）
江苏省自然科学基金资助项目（BK2006058）
关键词
大肠肿瘤
侵袭
基因转染
Colorectal neoplasms
Invasion
Gene transfection
