CAJ | 학술논문

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10 ng/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48 h,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10 h.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.
목적관찰내피세포표면주요조직상용성복합체-Ⅰ류상관련A(MICA)분자재NK세포대기살상과정중적작용,병탐토기가능적궤제.방법용10 ng/ml MICA중조항원대실험조인제정맥내피세포(HUVEC)진행유도배양48 h,대조조가입등량린산염완충액(PBS).용류식세포의(FCM)검측내피세포표면MICA분자적표체정황.사용면역자주방법급CD56양성분선시제합분선NK세포,병장NK세포여유도배양후적HUVEC공동배양10 h.용형광소진행염색,재형광현미경하관찰사망화존활HUVEC세포수량,계산NK세포대내피세포살상효솔,용ELISA법측정세포상청중적간우소-γ(IFN-γ)화천공소수평.결과실험조HUVEC세포표면MICA분자표체솔위(32±5.6)%,대조조위(6.0±2.4)%.NK세포대실험조HUVEC세포적살상효솔위(35.5±6.4)%,현저고우대조조적살상효솔(12.6±3.2)%,(P=0.0087);실험조HUVEC세포상청IFN-γ화천공소수평현저고우대조조,량조차이균유통계학의의(P=0.0025,P=0.0078).결론내피세포표면적MICA분자표체증가능구제고NK세포대내피세포적살상효솔;IFN-γ화천공소가능시발휘세포독작용적활성물질.
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.