锚定蛋白CD59与接头分子Cbp在T细胞上的定位及功能研究

Localization and function of CD59 and Cbp in T lymphocytes

目的研究CD59、Src羧基末端激酶结合蛋白(Cbp)在细胞膜的定位,及其在细胞活化及增殖中的相互作用。方法应用免疫荧光细胞化学技术,通过荧光显微镜分析系统对CD59、Cbp的定位进行了分析;Jurkat细胞转染pSUPER-siCD59重组质粒,并行RT-PCR和Western blot法检测转染细胞中CD59的表达及蛋白酪氨酸激酶癌基因家族(fyn)磷酸化水平。利用MTT比色法检测转染各组细胞的增殖效应。结果 CD59、Cbp主要分布在细胞膜上。转染pSUPER-siCD59重组质粒的Jurkat细胞中CD59表达量减少,磷酸化的fyn含量也随之减少,细胞增殖能力也低于其他各组。结论 CD59是一种膜蛋白,在细胞活化及增殖中与Cbp分子共同发挥作用。

Objective To investigate the distributions of GPI-anchored protein CD59 and C-terminal $rc kinase-binding protein （Cbp） in cell membrane of T lymphocytes and the roles in cell activation and proliferation. Methods The locations of CD59 and Cbp were observed under a fluorescence microscope with the immunofluorescence cytochemistry. The Jurkat cells were transfected with the recombinant plasmid pSUPER-siCD59 using the electric transfection method. Using RT-PCR and Western blotting, we detected the expression of CD59 and the phosphorylation level of protein-tyrosine kinase oncogene fam- ily （fyn） in different groups. Moreover, the cell proliferation activity was measured by M＇IF assay. Results The immunofluo- rescence cytochemistry showed that CD59 and Cbp were mainly distributed in cell membrane. Compared with other groups, the quantitative RT-P＇CR and Western blotting showed that the expression of CD59 was reduced, and along with it the phos- phorylation level of fyn decreased in pSUPER-siCD59 group, and MTT assay revealed that its cell proliferation was inhibited （P〈0.05）. Conclusion CD59 was a type of membrane-bound protein, CD59 and Cbp played synergistic roles in cell activation and proliferation.