CAJ | 학술논문

目的探讨自然杀伤细胞(NK)参与脑缺血后损伤的作用机制.方法 ①体内实验:免疫荧光检测永久性大脑中动脉栓塞(pMCAO)小鼠模型脑组织中NK细胞浸润情况以及干扰素(IFN)-γ表达情况;流式细胞术检测pMCAO小鼠模型脑中NK细胞浸润数目和IFN-γ表达;腹腔注射NK细胞中和抗体,流式细胞术检测pMCAO小鼠12 h脑内NK细胞浸润情况.②体外实验:建立体外血脑屏障并加入NK细胞和荧光标识的牛血清白蛋白(BSA-FITC),氧糖剥夺(OGD)实验6h后荧光分光光度计检测血脑屏障通透性;将NK细胞与小鼠神经细胞共培养,并加入IFN-γ阻断剂,OGD6h后流式细胞术检测神经细胞凋亡.结果 ①体内实验示pMCAO小鼠脑中有NK细胞浸润和IFN-γ表达,与非缺血侧相比,缺血侧的NK细胞浸润显著增多并于12 h达到高峰(P12h＜0.001);脑缺血组织中浸润的NK细胞表达IFN-γ与非缺血侧相比增多(P12h＜0.001、P24h＜0.01、P96h ＜0.05);清除体内NK细胞后,pMCAO小鼠脑内NK细胞浸润显著减少(P＜0.001),脑内IFN-γ表达显著下降(P ＜0.001).②体外实验示OGD条件下,NK细胞组与对照组相比,BSA-FITC渗透显著增多(P＜0.01);NK细胞可促进神经细胞死亡(P＜0.01).结论 NK细胞参与脑缺血损伤过程;NK细胞通分泌IFN-γ加重血脑屏障破坏和神经细胞损伤.
목적 탐토자연살상세포(NK)삼여뇌결혈후손상적작용궤제.방법 ①체내실험:면역형광검측영구성대뇌중동맥전새(pMCAO)소서모형뇌조직중NK세포침윤정황이급간우소(IFN)-γ표체정황;류식세포술검측pMCAO소서모형뇌중NK세포침윤수목화IFN-γ표체;복강주사NK세포중화항체,류식세포술검측pMCAO소서12 h뇌내NK세포침윤정황.②체외실험:건입체외혈뇌병장병가입NK세포화형광표식적우혈청백단백(BSA-FITC),양당박탈(OGD)실험6h후형광분광광도계검측혈뇌병장통투성;장NK세포여소서신경세포공배양,병가입IFN-γ조단제,OGD6h후류식세포술검측신경세포조망.결과 ①체내실험시pMCAO소서뇌중유NK세포침윤화IFN-γ표체,여비결혈측상비,결혈측적NK세포침윤현저증다병우12 h체도고봉(P12h＜0.001);뇌결혈조직중침윤적NK세포표체IFN-γ여비결혈측상비증다(P12h＜0.001、P24h＜0.01、P96h ＜0.05);청제체내NK세포후,pMCAO소서뇌내NK세포침윤현저감소(P＜0.001),뇌내IFN-γ표체현저하강(P ＜0.001).②체외실험시OGD조건하,NK세포조여대조조상비,BSA-FITC삼투현저증다(P＜0.01);NK세포가촉진신경세포사망(P＜0.01).결론 NK세포삼여뇌결혈손상과정;NK세포통분비IFN-γ가중혈뇌병장파배화신경세포손상.
Objective To research the mechanism of NK participating in brain ischemia.Methods ①Experiments in vivo:NK infiltration and IFN-γ in pMCAO mouse brain were detected by immunofluorescence; NK infiltration and IFN-γexpression in pMCAO brains were detected by flowcytometry; NK cells infiltration was detected by flowcytometry after NK cells depletion.②Experiments in vitro:Establish B.B.B.in vitro; B.B.B.in vitro were separated into Control,NK cells,BSA-FITC were added to the upper chamber of transwell,after OGD 6 h,permeability was detected by spectrofluorometer; Culturing NK cells together with neural cells and adding with IFN-γneutralizing antibody,and apoptosis experiment was detected by flowcytometry.Results ①Experiments in vivo:NK infiltration were higher in ischemia-hemisphere,and got a peak at 12 h(P12 h ＜0.01); and NK/IFN-γ cells infiltration were higher in ischemia-hemisphere (P12 h ＜ 0.001,P24 h ＜0.01,P96h ＜0.05);After NK cells depletion,NK cells infiltration and IFN-γ expression both obviously decreased(P ＜ 0.001).②Experiments in vitro:Compared with group Control,group N K has a higher B.B.B.permeability (P ＜ 0.01) ; NK cells increase neural dead cells percentage (P ＜ 0.01).Conclusion:NK cells plays a important role during brain ischemia; NK cells can promote B.B.B.permeability and neural cells death after cerebral ischemia via IFN-γ.