CCR7重组慢病毒的构建及其表达对结肠癌细胞侵袭的影响

摘要

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.%目的 构建含有CCR7的重组基因工程慢病毒,并转导结肠癌HT29细胞,高效表达CCR7蛋白,并研究趋化因子受体CCR7的表达在人结肠癌HT29细胞体外转移中的作用.方法 提取HT29细胞的RNA,RT-PCR扩增CCR7基因,克隆至慢病毒载体pLVX-acGFP-N1,形成重组慢病毒载体pLVX-acGFP-CCR7,转染293T细胞,得到慢病毒LV-CCR7,转导慢病毒LV-CCR7至HT29细胞,经puromycin筛选10 d.提取细胞的RNA,QPCR检测CCR7基因相对表达量的变化.划痕和Transwell小室实验分别检测HT29细胞和CCR7过表达细胞迁移和侵袭能力的差异.Western blotting检测CCR7蛋白和MMP-9蛋白相对表达量.结果 酶切和测序都表明CCR7克隆至慢病毒载体,慢病毒成功转导至HT29细胞,QPCR检测HT29细胞中CCR7表达升高了15.4倍,Western blotting检测CCR7蛋白表达上升了4.7倍.CCR7过表达的HT29细胞向划痕处爬行的速度明显快于HT29细胞对照组;过表达CCR7的HT29细胞透过膜的数量高于普通HT29细胞.过表达CCR7的HT29细胞与细胞侵袭转移相关的基质金属蛋白酶表达量显著高于普通HT29细胞.结论 利用含有CCR7的重组基因工程慢病毒成功构建了高效表达CCR7蛋白的结肠癌HT29细胞.研究发现高效表达的CCR7能促进结肠癌HT29细胞的体外迁移侵袭能力,增强了基质金属蛋白酶的表达.