摘要
目的观察过表达MHC Ⅰ类相关抗原A(MICA)的K562细胞,体外经诱导凋亡后,对树突状细胞(DC)吞噬功能的影响。方法首先利用脂质体介导的基因转染技术和G418筛选过程,建立稳定表达MICA的K562细胞(K562-MICA)。其次分别取K562、K562-MICA细胞经CFSE标记,并用丝裂霉素C诱导凋亡,与单核细胞系THP1或外周血单核细胞来源的DC共孵育过夜,流式细胞术检测2种细胞吞噬凋亡小体的活性。同时检测THP1细胞表面相关活化性受体的表达,以及DC表面NKG2D受体的情况。最后,在细胞共培养体系中加入NKG2D抗体,观察DC吞噬活性的变化。结果 K562-MICA细胞可刺激THP1细胞上调CD86和MICA的表达,但对HLA-DR及NKG2D的表达无明显影响。与K562细胞相比,来自K562-MICA的凋亡小体更容易被DC吞噬。K562-MICA凋亡小体可诱导DC上调NKG2D表达,NKG2D抗体具有显著拮抗DC吞噬功能的活性。结论 MICA过表达于K562细胞后具有促进DC吞噬功能的活性,这种活性依赖于DC表面NKG2D的表达。
Objective To observe how apoptosed K562 cells with over-expression of MHC class I chain-related protein A （MICA） affects phagocytic function of dendritic cells. Methods At first a K562 cell line with ectopic MICA expression, called K562-MICA, was generated by gene trans- fection and G418 screen. Both K562 and K562-MICA cells were stained with fluorescent CFSE, trea- ted with mitomycin C, and then co-cultured with THPI cells or dendritic cells derived from peripheral blood monocytes overnight. Phagocytic activities were evaluated through detection of percentage of ap- optotic cells by flow cytometry. Meanwhile, some activating receptors on THP1 cells and NKG2D ex- pression on DC were measured by flow cytometry. Finally NKG2D neutralizing antibody was added to cell co-culture system to observe whether phagocytosis of DC would be varied correspondingly. Results K562-MICA cells stimulated THP1 cell to enhance expression of CD86 and MICA, but had no effects on HLA-DR and NKG2D expression. Compared with K562 cells, apoptotic bodies from K562-MICA ceils were more susceptible to be uptake by DC. Apoptosed K562-MICA cells induced DC to increase NKG2D expression. In addition, NKG2D antibody could significantly inhibit phagocytosis of DC. Conclusion MICA over-expression on K562 cells promoted phagocytic function of DC, and the function depended on NKG2D expression on DC.
出处
《实用临床医药杂志》
CAS
2012年第22期14-17,共4页
Journal of Clinical Medicine in Practice
基金
国家自然科学基金(81172785
30671917)
江苏省自然科学基金(BK2011449
BK2008215)
江苏省教育厅大学生科技创新基金
