摘要
目的:探讨细胞因子CC配体2(CCL-2)对急性白血病细胞THP-1迁移和侵袭的影响及机制。方法:采用慢病毒包装的方法将表达质粒PLVX-CCL2转染THP-1细胞,用RT-PCR检测CCL-2 RNA水平的表达,Western blot及ELISA法验证蛋白水平的变化,应用Transwell迁移和侵袭实验方法分别检测CCL2重组蛋白及THP-1自身高表达CCL2对THP-1细胞迁移和侵袭能力的影响,应用迁移相关细胞因子的PCR array法探讨可能的机制。结果:RT-PCR和Western blot方法证实成功构建过表达CCL2的THP-1-CCL2细胞。ELISA法检测显示,THP-1-CCL2培养上清中CCL2蛋白水平显著增高;高表达CCL2的转染组细胞在Trans-Matrigel实验中,其上室CCL2蛋白浓度显著高于下室(P<0.001),同时穿膜能力下降,但迁移能力没有明显改变,与对照组比较,分别为(1.702±0.537 vs0.520±0.255)(P=0.026)和(9.293±0.302 vs 9.187±0.526)(P=0.77),增加Trans-Matrigel下室的CCL2浓度,可以增强THP-1细胞的迁移能力和侵袭能力。迁移RT2 profiler PCR array显示,CCL2重组蛋白处理THP-1细胞后,与对照组相比CCL2、EPX、SPP1、CX3CL1和CXCL13的表达上调(>2倍)。结论:CCL2通过白血病细胞THP-1的自分泌多种炎性趋化因子而调控其迁移和侵袭。
Objective： To investigate the effects and mechanism of CCL2 on the migration and invasion of human leukemia cell THP-1. Methods： The CCL2 gene was recombined with the transfer plasmid-PLVX and transfected into THP-1 cells. The CCL2 expression at RNA level was detected by RT-PCR,the CCL2 expression at protein level was determined by Western blot and ELISA,the influence of overexpression of CCL2 recombinant protein and THP-1 cells on the migration and invasion ability of THP-1 cells was analyzed by transwell migration and invasion tests,the PCRarray of migration-related cytokines was used to clarify the patential mechanism. Results： With the Trans-Matrigel assay,the concentration of CCL2 in THP-1 transfected with CCL2 in the upper cells was higher than that in the lower cells,meanwhile,the invasion ability of CCL2-transfected THP-1 cells decreased. Increasing recombinant protein of CCL2（ rpCCL2） in the lower cells promoted migration of THP-1 cells. Migration RT2 profiler PCR array showed that the cells treated with rpCCL2 had higher levels of expression of CCL2,EPX,SPP1,CX3 CL1 and CXCL13,as compared with control group. Conclusion： CCL2 affects the migration and invasion of THP-1 by autosecreting a series of inflammatory chemokines.
出处
《中国实验血液学杂志》
CAS
CSCD
北大核心
2018年第1期16-20,共5页
Journal of Experimental Hematology
基金
国家自然科学基金面上项目(81170513
81370627)
江苏省临床重点专病课题(BL2013014)
苏州市课题(LCZX201507
SZZX201504
SZS201615)
作者简介
通讯作者：胡绍燕，教授．E-mail：hsy139@126．com
