过表达白细胞介素8促进乳腺癌BT549细胞迁移  

Overexpression of IL-8 promotes migration of BT549 breast cancer cells

作　　者：邓芳[1] 王静[1] 范梦恬 郭杨柳 李亚[1] 施琼[1] 

机构地区：[1]重庆医科大学检验医学院临床检验诊断学教育部重点实验室,重庆400016

出　　处：《细胞与分子免疫学杂志》2016年第5期585-589,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(31200971);重庆市教委科学技术研究项目(KJ120327)

摘　　要：目的构建含有白细胞介素8(IL-8)基因的重组腺病毒,并观察其对BT549乳腺癌细胞增殖、细胞周期以及迁移能力的影响。方法以143B细胞c DNA为模版,PCR扩增IL-8基因,与穿梭质粒p Ad Track-TO4连接,构建重组穿梭质粒p Ad Track-TO4-IL-8,由PmeⅠ线性化并电转入Ad Easier细菌中,获得重组腺病毒质粒p Ad IL-8,PacⅠ酶切经LipofectamineTM2000介导转入HEK293细胞进行包装扩增,检测滴度;反转录PCR检测BT549细胞中IL-8 mRNA的水平,ELISA检测BT549细胞培养上清液中IL-8的水平;流式细胞术检测细胞周期情况;MTT法检测细胞增殖能力;划痕愈合实验检测细胞迁移能力。结果PCR以及测序证实p Ad Track-TO4-IL-8构建成功;p Ad IL-8经酶切证实重组正确;重组腺病毒Ad IL-8感染BT549细胞,可高表达IL-8。过表达IL-8的BT549细胞迁移能力增强,但其增殖活性无明显变化、将细胞周期阻滞于S期。结论过表达IL-8可促进BT549细胞的迁移。Objective To construct a recombinant adenovirus vector containing IL-8 gene and observe its effect on the proliferation, cell cycle and migration of BT549 breast cancer cells. Methods IL-8 gene was amplified by PCR using the cDNA from 143B bone sarcoma cells and inserted into shuttle plasmid pAdTrack-TO4. The recombinant shuttle plasmid pAdTrack-TO4-IL-8 was digested by Pine I and then transformed to AdEasier competent cells. The obtained recombinant adenovirus plasmid pAdlL-8 was digested by Pac I, and then transfected to HEK293 cells for package and amplification by LipofectamineTM 2000. The titer was tested by dilution assay. The expression of IL-8 mRNA and protein in BT549 cells was detected by reverse transcription PCR and ELISA, respectively. Effect of IL-8 overexpression on proliferation, cell cycle and migration in BT549 cells was respectively investigated by MTT assay, flow cytometry and wound-healing test. Results PCR and DNA sequence analysis verified the recombinant shuttle plasmid pAdTrack-TO4-1L-8. Restriction enzymes Pac I confirmed the recombinant adenovirus plasmid pAdlL-8. IL-8 was overexpressed in BT549 cells after AdlL-8 infection. Overexpression of IL-8 promoted BT549 cell migration and arrested the cell cycle in the S phase, but it made no significant difference in the proliferation of BT549 cells. Conclusion IL-8 overexpression can promote migration of BT549 breast cancer cells.

关 键 词：腺病毒 白细胞介素8(IL-8) BT549细胞 迁移 

分 类 号：R392.11[医药卫生—免疫学] R737.9[医药卫生—基础医学]