摘要
目的 探讨Transgelin-2过表达对结肠癌细胞侵袭转移的影响及其机制.方法 反转录-聚合酶链反应（RT-PCR）和Western blot技术检测5种结肠癌细胞株中Transgelin-2的mRNA和蛋白表达,筛选出Transgelin-2低表达细胞株SW480;以MegaTran 1.0试剂进行质粒转染,建立Transgelin-2过表达的结肠癌细胞模型;Transwell法检测Transgelin-2过表达对结肠癌细胞迁移侵袭能力的影响;激光共聚焦显微镜、透射电镜分别观察Transgelin-2过表达结肠癌细胞微丝的变化;纤维状肌动蛋白（F-actin）体外结合共沉淀实验验证Transgelin-2与F-actin的相互作用;激光共聚焦显微镜观察Transgelin-2与F-actin在细胞中的定位.结果 Transgelin-2过表达组的迁移细胞数在15 h[（207.27±62.26）个]、24 h[（179.00±32.08）个]均明显多于对照组[（114.13±29.17）个;（95.07±33.18）个;P＜0.05]及转染空载体组[（120.33±26.40）个;（95.93±28.66）个;P＜0.05];细胞中F-actin与单体球状肌动蛋白（G-actin）的相对含量发生明显变化,两者的荧光强度比值（18.7±8.6）较对照组（6.2±7.2）、空载体组均显著增加（8.8±4.0,P＜0.05）,提示发生微丝重塑;透射电镜观察发现过表达Transgelin-2的结肠癌细胞F-actin明显增多;F-actin体外结合共沉淀实验显示Transgelin-2与肌动蛋白存在体外的相互作用;激光共聚焦显微镜免疫荧光检测显示Transgelin-2与F-actin存在体内的共定位.结论 Transgelin-2可能通过参与调节微丝的重塑促进结肠癌细胞的迁移侵袭.
Objective To study the potential mechanism of Transgelin-2 over-expression enhanceing invasion and migration of colon cancer cells.Methods Reverse transcriptase-polymerase chain reaction （RT-PCR） and Western blotting were used to observe mRNA and protein expression of Transgelin-2 in 5 colorectal cancer （CRC） cell lines,and the cell lineSW480 with lower Transgelin-2 expression was selected.Transient transfection was performed to establish over-expression of Transgelin-2 cell line.The effect of Transgelin-2 over-expression on the ability of migration and invasion of colon cancer cells was detected by Transwell method.The microfilament change in colon cancer ceils with Transgelin-2 over-expression was observed by Laser scanning confocal microscope and transmission electron microscope.F-actin coprecipitation was used to validate the interaction between Transgelin-2 and F-actin in vitro.The cellular location of Transgelin-2 protein and F-actin was observed by immunocytochemistry.Results The number of migrating cells with Transgelin-2 over-expression was significantly increased as compared with control group and empty vector transfection group [15 h：（207.27 ± 62.26） cells vs.（114.13 ±29.17） cells and （120.33 ±26.40） cells;24 h：（179.00 ±32.08） cells vs.（95.07 ±33.18） cells and （95.93 ±28.66） cells,P 〈0.05 for all].The fluorescence intensity of F-actin and G-actin in colon cancer cells with Transgelin-2 over-expresion was significantly higher than that in control group and empty vector transfection group （18.7 ±8.6 vs.6.2 ±7.2 and 8.8 ±4.0）,suggesting microfilament remodeling occurred.Transmission electron microscopy demonstrated that F-actin levels were significantly increased in colon cancer cells with Transgelin-2 over-expression.Colocalization of Transgelin-2 and F-actin was noted in vivo by immunocytochemistry.Transmission electron microscopy demonstrated that there was interaction of Transgelin-2 and F-actin in vitro.Conclusion Transgelin-2 may enhance migration a
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2016年第9期2154-2157,共4页
Chinese Journal of Experimental Surgery
基金
北京大学人民医院研究与发展基金（RD-2014-14）
