慢病毒介导的KLF5转染对RKO细胞增殖和侵袭的影响  

Effects of lentivirus vector-mediated Kruppel-like factor 5 transfection on proliferation and metasta- sis of RKO cells

作　　者：赖东明[1] 周泉波[1] 李文滨[1] 褚忠华[1] 曾育杰[1] 陈双[1] 杨一增[1] 

机构地区：[1]中山大学孙逸仙纪念医院胃肠外科,广州510120

出　　处：《中华实验外科杂志》2012年第8期1556-1558,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（81072043、81071761）;广东省自然科学基金资助项目（10151008901000071）;广东省医学研究基金资助项目（A2010163）

摘　　要：目的构建Kruppel—likefactor5（KLF5）慢病毒载体并转染人结肠癌RKO细胞中，观察KLF5对结肠癌细胞RKO生物学行为的影响。方法采用逆转录-聚合酶链反应（RT—PCR）从人小肠黏膜中扩增出KLF5基因编码区1374bp的片段，随后将扩增KLF5片段插入慢病毒转移载体pCDH-CMV-KLF5-EFl-copGFP，构建KLF5-pCDH—CMV—KLF5-EFl-copGFP。在脂质体介导下将构建成功的重组慢病毒转染人胚肾细胞株（293T）包装生产慢病毒，测定病毒滴度，感染结肠癌RKO细胞。RT—PCR和Westernblot法分别检测KLF5mRNA和蛋白的表达。随后将实验分为空白对照组和实验转染组进行实验。细胞计数试剂盒（CCK-8）法检测转染前后RKO细胞增殖的变化以及Tr．answell实验检测转染前后细胞增殖和侵袭能力的变化。结果成功合成KLF5-pCDH—CMV—KLF5-EFl-copGFP并转染入RKO细胞中。RT—PCR以及Westernblot结果提示与对照组比较，KLF5-pCDH—CMV-KLF5-EFl-copGFP成功在RKO细胞中得到合成和表达。同时高表达的KLF5可以明显抑制RKO细胞的增殖（P〈0．05）。另外KLF5可以明显抑制RKO细胞的迁移能力（50．26±2．17比25．12±2．27，t=17．66，P〈0．05）和侵袭能力（45．48±1．53比22．134-2．25，t=3．37，P〈0．05）。结论KLF5可以有效抑制结肠癌RKO细胞的增殖、迁移和侵袭。Objective To investigate the effects of Kruppel-like factor 5 （KLF5） on biological be- haviors of RKO cell line. Methods With total RNA extracted from human intestinal cells as the template, KLF5 gene was amplified by reverse transcription-polymerase chain reaction （RT-PCR） with primers de- signed according to the sequence of GenBank, and the product was inserted into the pCDH-CMV-KLF5- EFI-copGFP to construct KLF5 lentivirus vector. RKO cells were divided into two groups： blank control group, KLF5 group. The synthesized KLF5 lentivirus vector was transfected into RKO cells. Real-time quantitative PCR and Western blotting were used to detect the KLF5 mRNA and protein levels. The prolif- eration of RKO cells was measured by cell counting Kit-8 （ CCK-8 ） assay. The ability of motility and inva- sion of RKO cells was assessed by Transwell invasion assay. Results In the KLF5 group, the expression of KLF5 mRNA and protein was increased as compared with the blank control group （ P 〈 0. 05 ）. The overex- pression of KLF5 could significantly suppressed proliferation of RKO cells （ P 〈 0.05 ）. KLF5 could signifi- cantly inhibit the abilities of migration and invasion of RKO cells as compared with the blank control group （50.26±2.17vs25.12±2.27;45.48±1.53vs22.13±2.25） （P〈0.05）.Conclusion KLF5could regulate the biological behaviors of RKO cell and be used as a target to prevent the colon cancer.

关 键 词：结肠癌 慢病毒 Kruppel—like FACTOR 5 

分 类 号：R735[医药卫生—肿瘤]