Mac-1在白细胞内的走向及归宿的动态定量研究

Mac-1(CD11b/CD18), one of the most important adhesion molecules, expressed on the surface of leukocytes consists of two subunits, known as αsubunit and β subunit. It plays an essential role in migration, chemotaxis and phagocytosis of leukocyte. Although there has been many reports about how Mac-1 acts in the process of adhesion with endothelial cell, information about the role of Mac-1 in deadhesion process is limited, few studies have been reported about dynamic quantitative analysis of its localization, and there has been only one mode action for different agonists. The present study was designed to investigate the intracellular trafficking of Mac-1..First, BFP and YFP, mutants of GFP, were selected because of their distinctive wavelength, and separately tagged at the carboxyl terminus of αsubunit and at the amino terminus of βsubunit of Mac-1 as recombinant plasmids pCD11b-BFP and pYFP-CD18 to minimize the possibility of negative effects on leukocyte function and observe clearly how αand βsubunits combine together and separate from Mac-1 dimer. CHO cell line, with signal pathway for inflammation and without internal Mac-1, was preferenced as the target cell for co-transfection with recombinant plasmids pCD11b-BFP and pYFP-CD18.To document that Mac-1- fluorescent protein (Mac-1-FP) expressed in CHO had the same structure and function as wild type Mac-1, we observed the blue fluorescence and yellow fluorescence from Mac-1-FP by fluorescence microscope, proved by western blot that there existed the Mac-1 dimer consisting of CD11b-BFP and YFP-CD18, demonstrated that cytoplasm Mac-1 could translocate to the cell membrane when induced by PMA using flow cytometry, and assayed the change of adhesive rate between Mac-1 and its ligand ICAM-1 incubated with PMA. When all these were completed, construction and expression of fusion protein were considered to be successful.Then, with PE-conjugated anti-CD11b IgG was used instead of CD11b-BFP, confocal microscopic observation and immunohistochemistry analysis after blocking translation of Mac-1 by cycloheximide reveal the following results: (1) Mac-1-FP translocated to cell membrane and clustered 1h after PMA, internalized 2 hours after PMA, and fluorescence diminished partially, probably because of degradations; the fluorescence recycle at 24 hours when PMA was re-administered. Dynamic changes of translocation and endocytosis of Mac-1 activated by PMA corresponded to inflections of the adhesion rate and varity of adhesion molecules on cell surface established by flow cytometry. (2) Adhesion rate of Mac-1-CHO cell line rose with the clustering of Mac-1-FP within 4 hours of interaction with ICAM-1 coupled with magnetic beads, and then reduced after 8 hours when fluorescence of Mac-1-FP diminished. This finding was similar to endocytosis and degradation induced by PMA.Based on these initial data, trafficking of Mac-1 is translocation from cytoplasm to cell membrane and endocytosis resulting in degradation or recycle, corresponding to inflections of adhesion rate. Thus, it is possible that endocytosis of Mac-1 is one of the most important mechanisms responsible for leukocyte deadhesion.

分别构建GFP标记a亚基因、BFP标记B亚基的高效表达载体，共转染入U937细胞，采用激光共聚焦显微镜和图像处理系统，对静态Mac-1的细胞内分布及不同的激动剂作用下，由贮存池头拧⒆弧⒈涔埂⑼崖浜竽谕谭纸饣蛟倮玫榷蹋卸ㄎ欢垦芯浚舛∕ac-1的亲和力与粘附活性的变化，阐明不同的促炎、抗炎因子对这一动态变化的影响。