摘要
目的观察内皮细胞表面主要组织相容性复合体-Ⅰ类相关链A（MICA）分子在NK细胞对其杀伤过程中的作用，并探讨其可能的机制。方法用10ng／ml MICA重组抗原对实验组人脐静脉内皮细胞（HUVEC）进行诱导培养48h，对照组加入等量磷酸盐缓冲液（PBS）。用流式细胞仪（FCM）检测内皮细胞表面MICA分子的表达情况。使用免疫磁珠方法及CD56阳性分选试剂盒分选NK细胞，并将NK细胞与诱导培养后的HUVEC共同培养10h。用荧光素进行染色，在荧光显微镜下观察死亡和存活HUVEC细胞数量，计算NK细胞对内皮细胞杀伤效率，用ELISA法测定细胞上清中的干扰素-γ（IFN-γ）和穿孔素水平。结果实验组HUVEC细胞表面MICA分子表达率为（32±5．6）％，对照组为（6．0±2．4）％。NK细胞对实验组HUVEC细胞的杀伤效率为（35．5±6．4）％，显著高于对照组的杀伤效率（12．6±3．2）％，（P=0．0087）；实验组HUVEC细胞上清IFN-γ和穿孔素水平显著高于对照组，两组差异均有统计学意义（P=0．0025，P=0．0078）。结论内皮细胞表面的MICA分子表达增加能够提高NK细胞对内皮细胞的杀伤效率；IFN-γ和穿孔素可能是发挥细胞毒作用的活性物质。
Objective To observe natural killer cell（NK） cytotoxicity to endothelial cell mediated by major histocompatibility complex class I-related chain A （MICA） molecule on the surface of endothelial cell. Methods HUVECs, an endothelial line, of experimental group were cultured for 48 h induced by 10 ng/ml recombinant MICA antigen. Another part of HUVECs was added with equal of volume of phosphote buffer salution（PBS） and worked as control group. MICA molecule on endothelial cell surface was detected by flow cytometery （FCM）. NK cells were collected by using immumomagnetic beads （CD56 positive isolation kit） according to the manufacturer＇ s recommended conditions. NK cells and HUVECs induced by MICA antigen were cocultured for 10 h. All the cells were stained by fluoresceins acridine orange（AO） and ernidium bromide（EB）. The dead and alive HUVECs were measured under fluorescence microscope, respectively. Killing effect of NK cells was calculated by counting of dead cells among total cells. Levels of IFN-γ and perforin in culfure supernate were detected by ELISA. Results Expression of MICA molecule on endothelial cell surface of experimental group was （32± 5.6 ）% and control group was （6.0 ± 2.4）%. The killing effect of NK cells in experimental group was （ 35.5 ± 6.4 ） %, which was significant higher than that in control group, （ 12.6 ± 3.2 ） % （ P = 0.0087）. Levels of IFN-γ, and perforin in experimental group were significant higher than that of control group （P =0.0025,0.0078）. Conclusion Expression of MICA molecule on the surface of HUVECs can enhance NK cytotoxicity to HUVECs. IFN-γ and perforin might be involved in the killing effect.
出处
《国际免疫学杂志》
CAS
北大核心
2011年第1期69-72,共4页
International Journal of Immunology
基金
国家自然科学基金资助项目（30872530）
江苏省自然科学基金资助项目（BK2007056）
江苏省卫生厅重点人才资助项目（RC2007079）
