CAJ | 학술논문

目的：探讨脑缺血发生后 IP－10对NK细胞的趋化作用。方法体内实验：免疫荧光染色观察永久性大脑中动脉栓塞小鼠模型（ pMCAO）脑组织中NK细胞浸润情况以及IP－10、CXCR3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；RT－PCR检测pMCAO小鼠模型脑中IP－10表达。体外实验：建立体外血脑屏障；ELISA检测氧糖剥夺（oxygen－glucose deprivation， OGD）6 h后内皮细胞和神经细胞上清IP－10表达；将体外血脑屏障分成NK组、 NK＋IP－10 blockade组，上层加入BSA－FITC，OGD 6 h后荧光分光光度计检测血脑屏障通透性，流式细胞术检测NK细胞迁移数目；向纯化的NK细胞中分别加入IP－10（10 ng／mL）、IP－10（50 ng／mL）和IP－10（100 ng／mL），流式细胞术检测CXCR3表达几何平均荧光强度。结果 pMCAO小鼠模型脑中有NK细胞浸润且表达CXCR3； NK细胞浸润缺血侧多于非缺血侧，12 h达到高峰（ P＜0．05）后逐渐下降，96 h开始略有升高；pMCAO小鼠模型脑中有IP－10表达，并于12 h达到高峰（ P＜0．001）后逐渐下降。体外实验：OGD后神经细胞表达IP－10较内皮细胞明显增多（ P＜0．001）；与NK组相比，NK＋IP－10 blockade组NK细胞迁移数目明显减少（ P＜0．05）；NK细胞表达CXCR3水平随IP－10作用浓度升高而升高。结论①NK细胞参与了脑缺血损伤过程；②脑缺血发生时IP－10通过与NK细胞表面表达的CXCR3结合，趋化NK细胞进入脑组织，并存在剂量依赖性。
목적：탐토뇌결혈발생후 IP－10대NK세포적추화작용。방법체내실험：면역형광염색관찰영구성대뇌중동맥전새소서모형（ pMCAO）뇌조직중NK세포침윤정황이급IP－10、CXCR3표체정황；류식세포술검측pMCAO소서모형뇌중NK세포침윤수목；RT－PCR검측pMCAO소서모형뇌중IP－10표체。체외실험：건입체외혈뇌병장；ELISA검측양당박탈（oxygen－glucose deprivation， OGD）6 h후내피세포화신경세포상청IP－10표체；장체외혈뇌병장분성NK조、 NK＋IP－10 blockade조，상층가입BSA－FITC，OGD 6 h후형광분광광도계검측혈뇌병장통투성，류식세포술검측NK세포천이수목；향순화적NK세포중분별가입IP－10（10 ng／mL）、IP－10（50 ng／mL）화IP－10（100 ng／mL），류식세포술검측CXCR3표체궤하평균형광강도。결과 pMCAO소서모형뇌중유NK세포침윤차표체CXCR3； NK세포침윤결혈측다우비결혈측，12 h체도고봉（ P＜0．05）후축점하강，96 h개시략유승고；pMCAO소서모형뇌중유IP－10표체，병우12 h체도고봉（ P＜0．001）후축점하강。체외실험：OGD후신경세포표체IP－10교내피세포명현증다（ P＜0．001）；여NK조상비，NK＋IP－10 blockade조NK세포천이수목명현감소（ P＜0．05）；NK세포표체CXCR3수평수IP－10작용농도승고이승고。결론①NK세포삼여료뇌결혈손상과정；②뇌결혈발생시IP－10통과여NK세포표면표체적CXCR3결합，추화NK세포진입뇌조직，병존재제량의뢰성。
Objective To research the chemotactic effect of IP-10 on natural killer cells after cerebral ischemia .Methods Experiments in vivo: NK infiltration and IP-10 and CXCR3 ex-pression in pMCAO mouse brain were detected by immunofluorescence;NK infiltration in pM-CAO brains was detected by flowcytometry;IP-10 total expression level in brain were investiga-ted by RT-PCR.Experiments in vitro:Established blood brain barrier ( B.B.B.) in vitro;IP-10 expression of the supernatant from brain microvascular endothclial cell ( BMVEC ) and neu-ral cells after oxygen-gluoose depriuation ( OGD) were detected by ELISA; B.B.B.in vitro were separated into NK , NK+IP-10 blockade two groups , after OGD 6 h, NK cells migration counts were detected by flowcytometry;NK cells were cultured together with IP -10 ( 10 ng/mL), IP-10 (50 ng/mL), IP-10(100 ng/mL), then CXCR3 geomean.MFI value were de-tected by flowcytometry .Results Experiments in vivo:NK cells were found in pMCAO brain section and expressing CXCR 3 and IP-10; NK cells infiltration were higher in ischemia-hemi-sphere , and got a peak at 12 h ( P<0.05 );NK cells had anobviously increase by 12 h at in-filtration frequency (P<0.01 );IP-10 had a highest level at 12 h ( P<0.001 ) .Experiments in vitro:Neural cell had higher expression of IP-10 after OGD than BMVEC ( P <0.001 );Compared with group NK , group NK+IP-10 blockade had lower NK cells migration counts ( P<0.05 ) and permeability ( P<0.001 ) .Conclusion ①NK cells plays an important role dur-ing cerebral ischemia;②IP-10 can absorb NK cells across B .B.B.when ischemia happens by combined with its receptor CXCR 3 and has dose dependent .