β-1,4-半乳糖基转移酶Ⅰ在CD4~ +T_H细胞中的表达及对其黏附能力的影响

Expression ofβ-1,4-galactosyltransferaseⅠin CD4^+ T_H cells and its effect on cellular adhesion ability

目的研究β-1,4-半乳糖基转移酶Ⅰ(β-1,4-galactosyltransferaseⅠ,β-1,4-GalTⅠ)在CD4^+TH中的表达与定化以及β-1,4-GalTⅠ对CD4^+TH细胞黏附能力的影响。方法采用免疫磁珠阳性分选法分离纯化CD4^+TH细胞,通过RT-PCR、荧光免疫细胞化学法和激光共聚焦技术鉴定CD4^+ TH细胞中β-1,4-GalTⅠ的表达与定位;分别用rhIL-2、rhIL-12、rhIL-4等细胞因子组合诱导CD4^+TH细胞活化分化,通过半定量RT-PCR、FCM检测CD4^+TH细胞中β-1,4-GalTⅠ的表达变化,运用层黏连蛋白结合实验比较CD4^+TH细胞黏附能力的改变;用α-乳清蛋白干扰CD4^+TH细胞表面β-1,4-GalTⅠ的活性及底物特异性,用层黏连蛋白结合实验分析其对CD4^+TH细胞黏附能力的影响。结果CD4^+TH细胞表面和细胞浆内表达长型和短型β-1,4-GalTⅠ;rhIL-2活化的CD4^+TH细胞长型β-1,4-GalTⅠmRNA水平及细胞膜表面β-1,4-GalTⅠ表达水平、细胞黏附能力均高于未经活化的CD4^+TH细胞, rhIL-2^+rhIL-12诱导培养的CD4^+TH细胞增强更明显;CD4^+TH细胞长型β-1,4-GalTⅠmRNA水平及细胞膜丧面β-1,4-GalTⅠ表达水平均与CD4^+TH细胞黏附能力呈正相关;α-浮清蛋白干扰后CD4^+TH细胞黏附能力降低。结论β-1,4-GalTⅠ在CD4^+TH细胞中表达,且与细胞黏附能力密切相关,提示β-1,4-GalTⅠ可能在CD4^+TH细胞的黏附过程中发挥重要作用。

Objective To study the expression and localization of β-1,4-galactosyltransferase I （β-1,4- GaIT I ）in CD4^＋ helper T（TH ）lymphocytes and the effect of β-1,4-GalT I on adhesion ability of CD4^＋ TH ceils. Methods CD4^＋ TH ceils were isolated by CD4 positive immunomagnetic beads. The expression and localization of β-1,4-GAIT I in CD4^＋ TH cells was detected by RT-PCR, immunofluorescence and laser scanning confocal microscopy. Activation and differentiation of CD4^＋ TH ceils were introduced by cytokines such as rhIL-2, rhIL-12 and rhIL-4. The expression level of β-1,4-GalT I in CD4^＋ TH cells was measured by semi-quantitative RT-PCR and flow cytometry. The adhesion ability of CD4^＋ TH cells was compared by ceil adhesion assay, α-lactalbumin was used to interfere the activity and substrate specificity of ceil surface β-1,4-GalT I , then the influence of that on the adhesion ability of CD4^＋ TH ceils was analyzed by ceil adhesion assay. Results long and short β-1,4-GaiT I were expressed and localized on the cytomembrane and cytoplasm of CD4^＋ TH ceils. The expression level of long β- 1,4-GalTI and cell surface β-1,4-GalTI , and the adhesion ability of CD4^＋ TH ceils activated with rhIL-2 in- creased as compared with unactivated CD4＋ TH cells. The expression level of long β-1,4-GalT I and ceil surface β-1,4-GAIT I were both positively correlated with the adhesion ability of CD4^＋ TH ceils. The adhesion ability of CD4^＋ TH ceils decreased after the incubation with α-lactalbumin. Conclusion β-1,4-GalT I expression in CD4^＋ TH ceils is closely correlated with ceil adhesion ability, which suggests that β-1,4-GalT I may play an important role in the adhesion process of CD4^＋ TH cells.