摘要
目的:探讨过表达Tie2基因后单核细胞功能的变化及对内皮细胞增殖、迁移、小管形成的影响,初步研究TEMs(Tie2-expressing monocytes)促血管生成的机制。方法:建立过表达Tie2单核细胞系,荧光倒置显微镜下观察U937细胞的感染情况,RT-PCR、Western blot分别检测感染后U937细胞Tie2 mRNA和蛋白水平的表达情况;实时定量PCR检测感染后空载组NC-L.V-U937和实验组TEK-L.V-U937细胞mRNA水平表达的差异;ELISA检测NC-L.V-U937和TEK-L.V-U937细胞上清中细胞因子分泌的异同;增殖、迁移、小管形成实验分别观察TEK-L.V-U937细胞对内皮细胞增殖、迁移、小管形成的影响。结果:首次建立了转染Tie2基因的单核细胞系;RT-PCR及Western blot结果显示,与空载组NC-L.V-U937细胞相比,实验组TEK-L.V-U937细胞在mRNA和蛋白水平Tie2的表达显著增加,而NC-L.V-U937细胞几乎不表达Tie2;Real time-PCR结果显示,TEK-L.V-U937细胞IL-8的表达较空载组显著增加(P<0.01),IL-10的表达也有所上升(P>0.05);与空载组相比,TEK-L.V-U937细胞IL-8蛋白分泌明显增多(P<0.05),VEGFA蛋白分泌量也有上升的趋势(P>0.05),但两组细胞几乎都无EGF的分泌;TEK-L.V-U937的条件培养基刺激内皮细胞形成的小管数较多,IL-8中和抗体能抑制内皮小管的形成;NC-L.V-U937、TEK-L.V-U937饥饿培养上清对内皮细胞的迁移作用不明显(P>0.05);增殖的第3天,与空载组相比,TEK-L.VU937的条件培养基能显著地引起内皮细胞的增殖(P<0.05)。结论:过表达Tie2基因后显著地增加U937单核细胞系IL-8的表达,并进一步促进了内皮细胞的增殖与小管形成。
Objective：To explore gene expression of Tie2-overexpressed monocytes and investigate its effect on endothelial cell proliferation,migration and tubule formation.Methods：Tie2 over-expressed monocytes were established.Fluorescent inverted microscope was used to observe the infection of U937.The mRNA and protein expression of Tie2 was measured by RT-PCR and Western blot,respectively.The difference between NC-L.V-U937 and TEK-L.V-U937 in mRNA level was measured by real-time PCR.Detection of cytokine secretion of NC-L.V-U937 and TEK-L.V-U937 were analyzed by ELISA.Endothelial cell proliferation,migration and tubule formation experiment were used to detect the effect of TEMs on HUVEC.Results：Compared with NC-L.V-U937 cells,the mRNA and protein expression of Tie2 increased significantly in TEK-L.V-U937 cells.Compared with control group,TEK-L.V-U937 cells expressed higher level of IL-8 （P ＜ 0.01） and IL-10 expression also increased（P ＞ 0.05）.The secretion of IL-8 increased significantly in TEK-L.V-U937 cells （P ＜ 0.05）,VEGFA secretion also had a rising trend （P ＞ 0.05）,but both cells had no EGF secretion.Conditioned medium from TEK-L.V-U937 cells induced more EC activation than NC-L.V-U937 cells.IL-8 neutralizing antibody inhibited tubule formation.Conditioned medium from TEK-L.V-U937 and NC-L.V-U937 cells had no effect on endothelial cell migration （P ＞ 0.05）.Compared with control group,the conditioned medium from TEK-L.V-U937 could significantly promote endothelial cell proliferation on the third day （P ＜ 0.05）.Conclusion：Over-expressing Tie2 significantly increase the expression of IL-8 in U937 cells and promote endothelial cell proliferation and tubule formation.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2014年第2期156-161,共6页
Chinese Journal of Immunology
基金
国家自然科学基金资助项目(No.81072136)
