基于流式细胞术快速定量分析小鼠角膜组织中嗜中性粒细胞方法的建立

Protocol for the rapid quantitative analysis of neutrophils in mouse cornea by flow cytometry

目的建立一种基于流式细胞术快速定量分析小鼠角膜组织中嗜中性粒细胞的技术和方法。方法选取6~8周龄SPF级雌性C57BL/6小鼠15只,使用高尔夫样刀机械性刮除小鼠角膜上皮细胞层,生成直径2 mm的创面,在创伤后18 h切除带有完整角膜缘的小鼠角膜,采用胶原酶I和DNA酶联合消化法获得单细胞悬液,采用FACSCanto流式细胞分析仪画门技术分选角膜细胞中嗜中性粒细胞的数量。另取6只小鼠,应用随机数字表法分为创伤组和正常组,每组3只,使用抗CD45、Ly6G和CD11b荧光抗体进行角膜细胞染色,计数并比较未创伤和创伤角膜中嗜中性粒细胞的数量变化。结果建立流式细胞仪检测角膜组织中嗜中性粒细胞的分析流程。CD45+细胞占角膜组织所有细胞的比例为(20.93±1.72)%,在角膜CD45+细胞群中可分选出Ly6G+CD11b+双阳性嗜中性粒细胞群,Ly6G+和CD11b+细胞在CD45+细胞中所占比例分别为(75.50±3.25)%和(93.40±4.53)%,Ly6G+和CD11b+共阳性细胞占角膜组织CD45+细胞的比例为(67.33±2.80)%。创伤后18 h,角膜中角膜缘募集嗜中性粒细胞数量为(151.47±10.82)%,多于正常角膜的(15.36±1.02)%,差异有统计学意义(t=21.689,P<0.01)。结论流式细胞检测方法可快速、准确地定量分析创伤角膜中嗜中性粒细胞群,为进一步评价不同原因造成角膜炎症反应中嗜中性粒细胞的数量变化提供了一种快速定量分析方法。

Objective To provide a standard protocol for the rapid quantitative analysis of neutrophils in inflamed corneas with flow cytometry.Methods The corneal epithelium layer of 15 C57BL/6 mice(6-8 weeks old)was mechanically scraped off using a golf-like knife to generate a 2 mm wound region.The mouse corneas with intact limbus were cut out at 18 hours after abrasion.After mechanical shredding,the single cell suspension was obtained by collagenase I and DNase digestion.Then,the number of neutrophils in the corneal cells was sorted under the FACSCanto flow cytometer using the gate technique.Another 6 mice were taken and randomized into wounded group and normal group according to a random number table method,with 3 mice in each group.Corneal cell staining was performed using fluorescent-conjugated anti-mouse CD45,Ly6G,and CD11b antibodies.The number of neutrophils in the corneas of the two groups were enumerated and compared.The use and care of the animals complied with the Statement of the Association for Research in Vision and Ophthalmology(ARVO).The study protocol was approved by the Animal Ethics Committee of Medical College of Jinan University(No.JN-A-2002-01).Results A standard procedure for detecting neutrophils in the cornea by flow cytometry was established.The ratio of CD45+cells in the total corneal tissue cell population was(20.93±1.72)%.The Ly6G+and CD11b+double positive neutrophil population was sorted in the wounded corneal cell population.The ratios of Ly6G+and CD116+cells in the CD45+cells were(75.50±3.25)%and(93.40±4.53)%,respectively,and the ratio of the Ly6G+and CD11b+double positive neutrophils in the total number of CD45+cells was(67.33±2.80)%.In addition,the number of neutrophils recruited to the cornea at 18 hours after corneal abrasion was(151.47±10.82)%,which was higher than(15.36±1.02)%in the normal cornea(t=21.689,P<0.01).Conclusions Flow cytometry can quickly and accurately quantitatively analyze the neutrophil population in the wounded cornea.It provides a rapid quantitative analysis method to further evaluate the changes of neutrophils in corneal inflammation caused by different reasons.