国际医药卫生导报2017，Vol.23Issue(23)：3664-3669,6.DOI:10.3760/cma.j.issn.1007-1245.2017.23.005
CCR7重组慢病毒的构建及其表达对结肠癌细胞侵袭的影响
Influences of construction of recombinant lentivirus carrying CCR7 gene and CCR7 expression on invasion of colorectal carcinoma cells
摘要
Abstract
Objective To construct the recombinant lentivirus vector containing CCR7 gene and detect the expression of CCR7 in colorectal carcinoma HT29 cells and to investigate the effect of over-expressed CCR7 gene in colorectal carcinoma cell line HT29.Methods RNA was extracted from HT29 cells;and CCR7 gene was amplified by RT-PCR.CCR7 DNA fragment was cloned into the lentiviral vector pLVX-acGFP-N1 to construct recombinant pLVX-acGFP-CCR7.Then the pLVX-acGFP-CCR7 was transfected into 293T cells to obtain infectious lentiviruses LV-CCR7.HT29 cells were infected with LV-CCR7 and selected with puromycin for 10 days.The efficacy of transduction was assessed examining the expression of CCR7 gene by fluorescence detection,QPCR,and western blotting.The invasive capacity of HT29 cells was examined by wound healing assay and transwell assay.Furthermore,the expression of MMP-9 was examined by western blotting.Results The CCR7 gene was cloned into the lentiviral vector confirmed by restriction enzyme digestion and DNA sequencing analysis.The green fluorescence was detected in HT29 cells after being transfected by LV-CCR7.The relative mRNA and protein expression levels of CCR7 in HT29 cells were 15.4 times and 4.7 times the control group's,respectively.There were much more over-expressed CCR7 tumor cells migrated into the gap compared with normal HT29 cells;and over-expressed CCR7 HT29 cells exhibited more invasion than the normal HT29 cells.The expression of MMP-9 increased in over-expressed CCR7 HT29 cancer cells.Conclusions The recombinant lentivirus vector containing CCR7 gene was successfully constructed and the CCR7 gene could be over-expressed in HT29 cells which promoted the migration and invasive ability of colorectal carcinoma cells.关键词
CCR7基因/慢病毒/结肠癌/细胞迁移/细胞侵袭Key words
CCR7 gene/Lentivirus/Colorectal cancer/Cell metastasis/Cell invasion引用本文复制引用
林汉利,陈小欢,王晓玲,曾浩涛..CCR7重组慢病毒的构建及其表达对结肠癌细胞侵袭的影响[J].国际医药卫生导报,2017,23(23):3664-3669,6.基金项目
深圳市科创委科技创新项目(JCY20140416094330192) （JCY20140416094330192）
龙岗区科技发展资金项目(YLWS20150513163509153) （YLWS20150513163509153）
Project Supported by Innovation of Science and Technology Commission of Shenzhen Municipality (JCY20140416094330192) （JCY20140416094330192）
Project of Longgang District Science and Technology Plan (YLWS20140609120004346) （YLWS20140609120004346）
