中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2014年
9期
780-785
,共6页
张红敏%刘素素%陈国铭%柳慧%窦新岩%王素娟%李金%王丽娅
張紅敏%劉素素%陳國銘%柳慧%竇新巖%王素娟%李金%王麗婭
장홍민%류소소%진국명%류혜%두신암%왕소연%리금%왕려아
角膜%感染%中性粒细胞%疾病模型%动物%C57BL/6J鼠%BALB/c鼠%裸鼠
角膜%感染%中性粒細胞%疾病模型%動物%C57BL/6J鼠%BALB/c鼠%裸鼠
각막%감염%중성립세포%질병모형%동물%C57BL/6J서%BALB/c서%라서
Cornea%Infection%Neutrophil%Disease model%Animal%C57BL/6J%BALB/c%Nude
背景 以往人们通常认为角膜无血管、无髓系来源的免疫细胞存在.C57BL/6J小鼠、BALB/c小鼠和裸鼠是眼科免疫学基础研究的常用模型,这些小鼠的角膜是否存在天然免疫的关键细胞——中性粒细胞是值得关注的问题. 目的 研究实验室常用的C57BL/6J鼠、BALB/c鼠和裸鼠正常角膜的中性粒细胞分布特征及其与角膜创伤修复的关系,为相关研究提供依据. 方法 选取角膜正常、10 ～ 12周龄的SPF级雄性C57BL/6J鼠、BALB/c鼠和BALB/c背景的裸鼠各16只,取3种小鼠各8只制备成中央区相连的4瓣角膜铺片,并以角膜周边血管缘为界向内以3个同心圆分区.分别用Gr-1-FITC抗体和CD31/PECAM-1-PE抗体对角膜中性粒细胞和血管进行免疫荧光染色,用免疫荧光显微镜和AR软件测量角膜血管面积并计数中性粒细胞.选取3种小鼠各8只制备角膜创伤模型,用无菌手术刀片以角膜中央为中心做十字划痕,深度至角膜前弹力层.创伤后即刻(0 h)、12h、24 h用2 g/L荧光素钠点眼,荧光显微镜下以AR软件计算不同时间点的创伤面积.于划痕后24 h取小鼠角膜制备铺片,用Gr-1-FITC抗体和CD31/PECAM-1-PE抗体行荧光染色,比较3种创伤小鼠角膜的血管分布、中性粒细胞的数量和分布情况.实验动物的饲养与使用均遵循美国视觉与眼科研究协会制定的科研动物使用规范. 结果 正常C57BL/6J小鼠、BALB/c小鼠和裸鼠角膜中性粒细胞总数分别为(1 733±237)、(353±96)和(1 601 ±223)个/角膜,BALB/c小鼠角膜中性粒细胞数明显少于C57BL/6J小鼠和裸鼠,差异均有统计学意义(P＜0.01).3种正常小鼠角膜缘均可见血管分布,BALB/c小鼠角膜缘血管面积明显小于C57BL/6J小鼠和裸鼠,C57BL/6J小鼠角膜缘血管面积明显大于裸鼠,差异均有统计学意义(P＜0.01).3种正常小鼠中性粒细胞的数量与血管面积均呈明显正相关(C57BL/6J:r=0.936,P=0.001;BALB/c:r =0.939,P=0.001;裸鼠:r=0.935,P=0.001).角膜划痕后24 h,C57BL/6J小鼠和裸鼠的创伤面积均明显大于BALB/c小鼠(均P=0.000),且以裸鼠的创伤面积最大.角膜划痕后C57BL/6J模型鼠、BALB/c模型鼠和模型裸鼠的角膜上中性粒细胞数目分别为(3 340±417)、(1 235±337)和(4 680±450)个/角膜,BALB/c模型鼠角膜上中性粒细胞数目明显少于C57BL/6J模型鼠和模型裸鼠,差异均有统计学意义(均P=0.000).3种小鼠角膜创伤面积与中性粒细胞数目均呈明显正相关(C57BL/6J小鼠:r=0.805,P=0.016;BALB/c小鼠:r=0.943,P=0.000;裸鼠:r=0.916,P=0.001). 结论 不同品系的正常小鼠角膜中性粒细胞分布的数目取决于角膜血管的分布和面积,角膜损伤后小鼠角膜中性粒细胞数目增加,角膜中性粒细胞数目较少的小鼠其角膜创伤修复速度较快,提示角膜血管网部位中性粒细胞的基础状态可能与角膜对感染的易感性以及感染后的早期反应有关.
揹景 以往人們通常認為角膜無血管、無髓繫來源的免疫細胞存在.C57BL/6J小鼠、BALB/c小鼠和裸鼠是眼科免疫學基礎研究的常用模型,這些小鼠的角膜是否存在天然免疫的關鍵細胞——中性粒細胞是值得關註的問題. 目的 研究實驗室常用的C57BL/6J鼠、BALB/c鼠和裸鼠正常角膜的中性粒細胞分佈特徵及其與角膜創傷脩複的關繫,為相關研究提供依據. 方法 選取角膜正常、10 ～ 12週齡的SPF級雄性C57BL/6J鼠、BALB/c鼠和BALB/c揹景的裸鼠各16隻,取3種小鼠各8隻製備成中央區相連的4瓣角膜鋪片,併以角膜週邊血管緣為界嚮內以3箇同心圓分區.分彆用Gr-1-FITC抗體和CD31/PECAM-1-PE抗體對角膜中性粒細胞和血管進行免疫熒光染色,用免疫熒光顯微鏡和AR軟件測量角膜血管麵積併計數中性粒細胞.選取3種小鼠各8隻製備角膜創傷模型,用無菌手術刀片以角膜中央為中心做十字劃痕,深度至角膜前彈力層.創傷後即刻(0 h)、12h、24 h用2 g/L熒光素鈉點眼,熒光顯微鏡下以AR軟件計算不同時間點的創傷麵積.于劃痕後24 h取小鼠角膜製備鋪片,用Gr-1-FITC抗體和CD31/PECAM-1-PE抗體行熒光染色,比較3種創傷小鼠角膜的血管分佈、中性粒細胞的數量和分佈情況.實驗動物的飼養與使用均遵循美國視覺與眼科研究協會製定的科研動物使用規範. 結果 正常C57BL/6J小鼠、BALB/c小鼠和裸鼠角膜中性粒細胞總數分彆為(1 733±237)、(353±96)和(1 601 ±223)箇/角膜,BALB/c小鼠角膜中性粒細胞數明顯少于C57BL/6J小鼠和裸鼠,差異均有統計學意義(P＜0.01).3種正常小鼠角膜緣均可見血管分佈,BALB/c小鼠角膜緣血管麵積明顯小于C57BL/6J小鼠和裸鼠,C57BL/6J小鼠角膜緣血管麵積明顯大于裸鼠,差異均有統計學意義(P＜0.01).3種正常小鼠中性粒細胞的數量與血管麵積均呈明顯正相關(C57BL/6J:r=0.936,P=0.001;BALB/c:r =0.939,P=0.001;裸鼠:r=0.935,P=0.001).角膜劃痕後24 h,C57BL/6J小鼠和裸鼠的創傷麵積均明顯大于BALB/c小鼠(均P=0.000),且以裸鼠的創傷麵積最大.角膜劃痕後C57BL/6J模型鼠、BALB/c模型鼠和模型裸鼠的角膜上中性粒細胞數目分彆為(3 340±417)、(1 235±337)和(4 680±450)箇/角膜,BALB/c模型鼠角膜上中性粒細胞數目明顯少于C57BL/6J模型鼠和模型裸鼠,差異均有統計學意義(均P=0.000).3種小鼠角膜創傷麵積與中性粒細胞數目均呈明顯正相關(C57BL/6J小鼠:r=0.805,P=0.016;BALB/c小鼠:r=0.943,P=0.000;裸鼠:r=0.916,P=0.001). 結論 不同品繫的正常小鼠角膜中性粒細胞分佈的數目取決于角膜血管的分佈和麵積,角膜損傷後小鼠角膜中性粒細胞數目增加,角膜中性粒細胞數目較少的小鼠其角膜創傷脩複速度較快,提示角膜血管網部位中性粒細胞的基礎狀態可能與角膜對感染的易感性以及感染後的早期反應有關.
배경 이왕인문통상인위각막무혈관、무수계래원적면역세포존재.C57BL/6J소서、BALB/c소서화라서시안과면역학기출연구적상용모형,저사소서적각막시부존재천연면역적관건세포——중성립세포시치득관주적문제. 목적 연구실험실상용적C57BL/6J서、BALB/c서화라서정상각막적중성립세포분포특정급기여각막창상수복적관계,위상관연구제공의거. 방법 선취각막정상、10 ～ 12주령적SPF급웅성C57BL/6J서、BALB/c서화BALB/c배경적라서각16지,취3충소서각8지제비성중앙구상련적4판각막포편,병이각막주변혈관연위계향내이3개동심원분구.분별용Gr-1-FITC항체화CD31/PECAM-1-PE항체대각막중성립세포화혈관진행면역형광염색,용면역형광현미경화AR연건측량각막혈관면적병계수중성립세포.선취3충소서각8지제비각막창상모형,용무균수술도편이각막중앙위중심주십자화흔,심도지각막전탄력층.창상후즉각(0 h)、12h、24 h용2 g/L형광소납점안,형광현미경하이AR연건계산불동시간점적창상면적.우화흔후24 h취소서각막제비포편,용Gr-1-FITC항체화CD31/PECAM-1-PE항체행형광염색,비교3충창상소서각막적혈관분포、중성립세포적수량화분포정황.실험동물적사양여사용균준순미국시각여안과연구협회제정적과연동물사용규범. 결과 정상C57BL/6J소서、BALB/c소서화라서각막중성립세포총수분별위(1 733±237)、(353±96)화(1 601 ±223)개/각막,BALB/c소서각막중성립세포수명현소우C57BL/6J소서화라서,차이균유통계학의의(P＜0.01).3충정상소서각막연균가견혈관분포,BALB/c소서각막연혈관면적명현소우C57BL/6J소서화라서,C57BL/6J소서각막연혈관면적명현대우라서,차이균유통계학의의(P＜0.01).3충정상소서중성립세포적수량여혈관면적균정명현정상관(C57BL/6J:r=0.936,P=0.001;BALB/c:r =0.939,P=0.001;라서:r=0.935,P=0.001).각막화흔후24 h,C57BL/6J소서화라서적창상면적균명현대우BALB/c소서(균P=0.000),차이라서적창상면적최대.각막화흔후C57BL/6J모형서、BALB/c모형서화모형라서적각막상중성립세포수목분별위(3 340±417)、(1 235±337)화(4 680±450)개/각막,BALB/c모형서각막상중성립세포수목명현소우C57BL/6J모형서화모형라서,차이균유통계학의의(균P=0.000).3충소서각막창상면적여중성립세포수목균정명현정상관(C57BL/6J소서:r=0.805,P=0.016;BALB/c소서:r=0.943,P=0.000;라서:r=0.916,P=0.001). 결론 불동품계적정상소서각막중성립세포분포적수목취결우각막혈관적분포화면적,각막손상후소서각막중성립세포수목증가,각막중성립세포수목교소적소서기각막창상수복속도교쾌,제시각막혈관망부위중성립세포적기출상태가능여각막대감염적역감성이급감염후적조기반응유관.
Background C57BL/6J,BALB/c and nude mice are commonly used models in laboratory study of eye immunology,and neutrophil is a natural immune barrier.Previous studies showed that there are no vessel and neutrophils in mouse cornea.Therefore,whether there are neutrophils,the key natural immune cells,in mouse cornea is worth studying.Objective This study was to illuminate the distribution traits of neutrophils in normal cornea in C57BL/6J mice,BALB/c mice and nude mice,and to investigate the relation between corneal neutrophil and wound repair.Methods Male SPF C57BL/6J,BALB/c and nude mice with a BALB/c background were enrolled in this study.The whole mount mouse cornea was prepared and divided into 3 concentric zones.Gr-1-FITC and CD31/ PECAM-1-PE antibodies were used to label the neutrophils and vessels.Corneal wound models were established using scratching method.Fluorescine sodium was used to observe the corneal wound area.The whole mount cornea was prepared and corneal vessels and neutrophils were quantified by Gr-1-FITC and CD31/PECAM-1-PE antibody staining,respectively.The use and care of the animals complied with the Statement of Association for Research in Vision and Ophthalmology (ARVO).Results The number of neutrophils was (1 733±237),(353±96) and (1 601±223)/cornea in C57BL/6J,BALB/c and nude mice,respectively,and the number of neutrophils in cornea was significantly reduced in BALB/c mice compared with C57BL/6J and nude mice (both at P＜0.01).The blood vessels were seen in limbal with the red fluorescence for CD31-PE antibody in three strains of mice,and vessel area was smaller in the BALB/c mice than those in C57BL/6J and nude mice (both at P＜0.01).The corneal vessel area was the largest in the C57BL/6J mice.Significantly positive correlations were found between the corneal vessel area and number of neutrophils in three strains of mice (C57BL/6J mice:r=0.936,P=0.001 ;BALB/c mice:r=0.939,P =0.001 ; nude mice:r =0.935,P =0.001).In 24 hours after corneal scratching,the wound areas were larger in the C57BL/6J mice and nude mice than those in the BALB/c mice (both at P =0.000),and the corneal wound area was the largest in the nude mice.The number of neutrophils was (3 340±417),(1 235±337) and (4 680 ±450)/cornea in the C57BL/6J,BALB/c and nude wound mice,showing significant differences between the BALB/c models and C57BL/6J models or nude models (all at P=0.000).In addition,positive correlations were seen between the corneal wound area and the number of neutrophils in all the three model groups (C57BL/6J models:r =0.805,P =0.016 ; BALB/c models:r=0.943,P=0.000;nude models:r=0.916,P=0.001).Conclusions The number of corneal neutrophils depends on the distribution and area of corneal vessels in three strains of mice.The number of neutrophils is associated with corneal wound healing.These results suggest that the distribution and number of neutrophils in cornea are associated with the infection susceptibility and early inflammatory response.
