CAJ | 학술논문

目的构建Kruppel-like factor 5(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1374 bp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-EF1-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Western blot法分别检测KLF5 mRNA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Transwell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-EF1 -copGFP并转染入RKO细胞中.RT-PCR以及Western blot结果提示与对照组比较,KLF5-pCDH-CMV-KLF5-EF1 -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22.13 ±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.
목적 구건Kruppel-like factor 5(KLF5)만병독재체병전염인결장암RKO세포중,관찰KLF5대결장암세포RKO생물학행위적영향.방법 채용역전록-취합매련반응(RT-PCR)종인소장점막중확증출KLF5기인편마구1374 bp적편단,수후장확증KLF5편단삽입만병독전이재체pCDH-CMV-KLF5 -Efl -copGFP,구건KLF5-pCDH-CMV-KLF5-EF1-copGFP.재지질체개도하장구건성공적중조만병독전염인배신세포주(293T)포장생산만병독,측정병독적도,감염결장암RKO세포.RT-PCR화Western blot법분별검측KLF5 mRNA화단백적표체.수후장실험분위공백대조조화실험전염조진행실험.세포계수시제합(CCK-8)법검측전염전후RKO세포증식적변화이급Transwell실험검측전염전후세포증식화침습능력적변화.결과 성공합성KLF5-pCDH-CMV-KLF5-EF1 -copGFP병전염입RKO세포중.RT-PCR이급Western blot결과제시여대조조비교,KLF5-pCDH-CMV-KLF5-EF1 -copGFP성공재RKO세포중득도합성화표체.동시고표체적KLF5가이명현억제RKO세포적증식(P＜0.05).령외KLF5가이명현억제RKO세포적천이능력(50.26±2.17비25.12 ±2.27,t=17.66,P＜0.05)화침습능력(45.48±1.53비22.13 ±2.25,t=3.37,P＜0.05).결론 KLF5가이유효억제결장암RKO세포적증식、천이화침습.
Objective To investigate the effects of Kruppel-like factor 5 ( KLF5 ) on biological behaviors 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 designed according to the sequence of GenBank,and the product was inserted into the pCDH-CMV-KLF5-EF1-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 proliferation of RKO cells was measured by cell counting Kit-8 (CCK-8) assay.The ability of motility and invasion 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 overexpression of KLF5 could significantly suppressed proliferation of RKO cells ( P ＜ 0.05 ).KLF5 could significantly inhibit the abilities of migration and invasion of RKO cells as compared with the blank control group ( 50.26 ± 2.17 vs 25.12 ± 2.27 ; 45.48 ± 1.53 vs 22.13 ± 2.25 ) (P＜0.05).Conclusion KLF5 could regulate the biological behaviors of RKO cell and be used as a target to prevent the colon cancer.