γ-synuclein基因真核表达载体的构建及其对结肠癌细胞SW1116体外侵袭转移潜能的影响  

Construction of γ-synuclein eukaryotic expression vector and its effect on invasion and metastasis of colon cancer cell line SW1116 in vitro

作　　者：叶青[1] 黄峰[1] 郑秋红[1] 王晓盈[1] 许扬梅[1] 龚福生[1] 黄丽洁[1] 

机构地区：[1]福建医科大学附属肿瘤医院腹部外科福建省肿瘤生物治疗重点实验,福州350014

出　　处：《中华胃肠外科杂志》2014年第1期36-40,共5页Chinese Journal of Gastrointestinal Surgery

基　　金：基金项目：国家自然科学基金青年科学基金（81101897）;福建省自然科学基金青年创新项目（2011J05061）

摘　　要：目的构建针对γ-synuclein基因的真核表达载体并观察其对结肠癌细胞sw1116体外侵袭及与人脐静脉内皮细胞（HUVEC）黏附能力的影响。方法从结肠癌细胞系HT29提取总RNA，经RT．PCR获得γ-synucleincDNA全长片段，经过酶切连接等反应定向克隆至真核绿色荧光蛋白表达载体pEGFP-C1上，以脂质体转染的方法将重组质粒转染至结肠癌细胞系SW1116，经G418筛选出稳定转染的细胞株。采用Transwell小室法检测细胞体外侵袭能力。将细胞接种于铺有HUVEC的96孔板中，酶标仪读取荧光强度来表示黏附细胞的相对数。结果成功构建pEGFP-γ-synuclein真核表达载体，经过筛选后可在SW1116细胞中稳定表达，且能在体外翻译出GFP-γ-synuclein融合蛋白质。转染γ-synuclein后，穿过Matrigel胶及Transwell小室膜的细胞数明显增加（198．4±20．7比98．8±13．2，P〈0．05），与HUVEC黏附的细胞数（荧光强度）亦显著增加（3．08±0．36比1．22±0．21，P〈0．05）。结论γ-synuclein可显著增强结肠癌细胞SW1116体外侵袭转移潜能。Objective To construct γ-synuclein gene eukaryotic expression vector, and to study its effect on the invasion of colon cancer cell line SW1116 and the adhesion between SW1116 and human umbilical vein endothelial cells （HUVECs） in vitro. Methods Total RNA was extracted from colon cancer cell line HT＇29 and the eDNA of γ-synuelein was amplified using RT-PCR. The digested fragment of eDNA coding sequence was linked to the eukaryotic expression vector pEGFP-C 1 containing the GFP gene. After identification by sequence analysis, the recombinant plasmid was transfected into colon cancer cell line SW1116 via lipofectamine. The stable cell line was selected with G-418. The invasion in vitro was tested by Transwell invasion chamber assay. HUVECs were previously seeded onto 96-well plates before SW1116 cells seeded, and fluorescence intensity of GFP was detected to represent the amount of adhesion cells by ELISA. Results Human γ-synuelein eukaryotic expression vector was successfully constructed, which was stably expressed in SW1116 cells and could translate the GFP-γ synuclein protein in vitro. γ-synuclein facilitated SW1116 cell passing through matrigel and filter membrane （198.4±20.7 vs. 98.8±13.2, P〈0.05） and elevated the adherence of SWl116 cells toHUVECs （3.08±0.36 vs. 1.22±0.21, P〈0.05）. Conclusion Expression of γ-synuelein can strengthen colon cancer cell SW1116 potentiality of invasion and metastasis in vitro.

关 键 词：结肠癌细胞 基因 γ-synuclein 侵袭 黏附 人脐静脉内皮细胞 

分 类 号：R735.3[医药卫生—肿瘤]