Inflammatory cytokine of basal and reflex tears analysed by multicytokine assay
Inflammatory cytokine of basal and reflex tears analysed by multicytokine assay
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DOI：
10.1136/bjo.2005.076737
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发表时间：
2006-01-01
影响因子：
4.1
通讯作者：
Sakamoto, T
中科院分区：
文献类型：
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作者：
Sonoda, S;Uchino, E;Sakamoto, T
方法招募正常志愿者23人，男11人，女12人，年龄22 ~ 44岁，平均28岁。所有受试者都没有眼部疾病的迹象。这项研究是在机构审查委员会的批准下进行的。在没有鼻腔刺激和注射药物或生命染料的情况下，通过毛细管流动从每只眼获得10-15 μl的基底泪液样本。每个样本于下午5点采集。未滴注麻醉滴剂。样本是从下半月板非创伤性收集的。随后，通过将应用棒插入参与者的鼻子来收集反射性泪液样本。根据制造商的说明，用CBA （BD Biosciences, San Diego， CA， USA）测量六种炎症分子白介素(IL)-1β、IL-6、IL-8、IL-10、IL-12p70和肿瘤坏死因子α (TNF-α)的含量。简单地说，泪液样品和细胞因子标准品混合后，在40 μl无菌纯净水中加入10 μl的样品或标准品，捕获抗体头试剂和检测抗体-藻红蛋白（PE）试剂各50 μl的混合物。随后将混合物在室温下孵育3小时，并在流式细胞术采集数据之前清洗以去除任何未结合的Ab-PE检测器试剂。使用FACScan流式细胞仪（Beckton Dickinson Immunocytometry Systems）进行双色流式细胞仪分析。数据采集和分析使用BD细胞仪头阵列软件。结果基底性和反射性泪液中IL-1β、IL-6、IL-10、IL-12p70和TNF-α的含量无显著差异。相比之下，每只眼反射性泪液中IL-8的浓度明显低于基底泪液（配对t检验，p< 0.01，图1）。为了阐明各细胞因子之间的相互关系，表1给出了两种不同细胞因子的比值。
MethodsTwenty three normal volunteers (11 males and 12 females, 22–44 years of age, average 28 years) were recruited for this study. None of the subjects had signs of ocular diseases. The study was performed with the approval of the institutional review board. The basal tear samples of 10–15 μl were obtained from each eye by capillary flow, with no nasal stimulation or previous instillation of drugs or vital dyes. Each sample was collected at 5 pm. No anaesthetic drops were instilled. The samples were collected non-traumatically from the inferior meniscus. Successively, reflex tear samples were collected by inserting application sticks into a participant’s nose. The amounts of six inflammatory molecules interleukin (IL)-1β, IL-6, IL-8, IL-10, IL-12p70, and tumour necrosis factor α (TNF-α), were measured by CBA (BD Biosciences, San Diego, CA, USA), according to the manufacturer’s instructions. Briefly, for the tear sample and cytokine standard mixture, 10 μl of sample or standard were added to 40 μl sterile purified water, a mixture of 50 μl each of capture Ab-bead reagent and detector Ab-phycoerythrin (PE) reagent. The mixture was subsequently incubated for 3 hours at room temperature, and washed to remove any unbound detector Ab-PE reagent before data acquisition using flow cytometry. A two colour flow cytometric analysis was performed using a FACScan flow cytometer (Beckton Dickinson Immunocytometry Systems). Data were acquired and analysed using BD cytometric bead array software.ResultsThe concentrations of IL-1β, IL-6, IL-10, IL-12p70, and TNF-α were not significantly different between basal and reflex tears. In contrast, the concentration of IL-8 was significantly decreased in reflex tears compared with basal tears in each eye (paired t test, p< 0.01, fig 1). In order to illuminate the inter-relation of each cytokine, the ratio of two different cytokines is shown in table 1.