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

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。

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 l0 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 recombinantlentivirus 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.