Sakata M, Sack R A, Sathe S, Holden B, Beaton A R
State University of New York, College of Optometry, Department of Biological Sciences, NY 10010, USA.
Curr Eye Res. 1997 Aug;16(8):810-9. doi: 10.1076/ceyr.16.8.810.8992.
To characterize the effects that mode of sampling and overnight eye closure have on the nature of caseinolytic activity recovered in tear fluid.
Reflex, open and closed (R, O and C) eye tear fluids were collected by microcapillary tubes or from the inferior formix by Schirmer strip. Microcapillary collected samples were centrifuged and recovered cells cytochemically characterized and probed by immunofluorescence microscopy, or alternatively extracted in acidic PBS. Tear supernatants, pellets and Schirmer strip extracts were subjected to casein zymography or SDS-PAGE and immunoprobed for plasmin/plasminogen. To identify caseinolytic activity, samples were immunoprecipitated with antibodies to plasmin/plasminogen or to elastase, and the immunoprecipitated materials were subjected to zymographic analysis.
Immunoblot assays revealed R and O samples contained low levels of plasminogen (approximately 1.1 micrograms/ml) and only trace levels of plasmin (< 0.1 ng/ml). Insufficient levels of caseinolytic activity were present to allow zymographic detection. Cytochemical analysis revealed that R and O pellets consisted almost exclusively of desquamated epithelium. Immunoblot analysis revealed that C fluid was associated with an increase in plasminogen and its partial conversion to plasmin (approximately 3.2 ng/ microliter), high molecular weight covalent complexes and degradative products. Zymographic analysis disclosed much greater caseinolytic activity than could be attributed to plasmin or its cleavage products. This consisted primarily of three bands (30-26 kDa) which were identified as polymorphonuclear leukocyte (PMN) cell elastase based on size and antigenicity. This is derived from PMNs recovered from the C pellet. Elastase could also be recovered from Schirmer strips from 90% of donors, provided that the strips were extracted in sample loading buffer. The activity was restricted to the portion of the strip that had been in contact with the ocular tissue.
The main source of caseinolytic activity in C fluid is elastase. This arises from PMNs that undergo recruitment, activation and degranulation in the C environment. In contrast, the elastase recovered in Schirmer strip extracts is derived from intact PMNs that adhere to the strip during sample collection. This would suggest that PMN cells undergo a low level of recruitment into the open eye environment.
描述采样方式和夜间闭眼对泪液中酪蛋白溶解活性性质的影响。
用微量毛细管或通过Schirmer试纸从下穹窿收集反射性、睁眼和闭眼（R、O和C）时的眼泪液。微量毛细管收集的样本进行离心，回收的细胞进行细胞化学特征分析并通过免疫荧光显微镜检测，或者在酸性磷酸盐缓冲盐水中提取。泪液上清液、沉淀和Schirmer试纸提取物进行酪蛋白酶谱分析或十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE），并针对纤溶酶/纤溶酶原进行免疫检测。为鉴定酪蛋白溶解活性，样本用抗纤溶酶/纤溶酶原或抗弹性蛋白酶抗体进行免疫沉淀，免疫沉淀的物质进行酶谱分析。
免疫印迹分析显示，R和O样本中纤溶酶原水平较低（约1.1微克/毫升），纤溶酶水平仅为痕量（<0.1纳克/毫升）。酪蛋白溶解活性水平不足以进行酶谱检测。细胞化学分析显示，R和O沉淀几乎完全由脱落的上皮细胞组成。免疫印迹分析显示，C液中纤溶酶原增加并部分转化为纤溶酶（约3.2纳克/微升），存在高分子量共价复合物和降解产物。酶谱分析显示，酪蛋白溶解活性比可归因于纤溶酶或其裂解产物的活性高得多。这主要由三条带（30 - 26千道尔顿）组成，根据大小和抗原性鉴定为多形核白细胞（PMN）弹性蛋白酶。这源自从C沉淀中回收的PMN。弹性蛋白酶也可从90%供体的Schirmer试纸中回收，前提是试纸在样品上样缓冲液中提取。活性仅限于试纸与眼组织接触的部分。
C液中酪蛋白溶解活性的主要来源是弹性蛋白酶。这源于在C环境中经历募集、激活和脱颗粒的PMN。相比之下，Schirmer试纸提取物中回收的弹性蛋白酶源自样本收集过程中粘附在试纸上的完整PMN。这表明PMN细胞在睁眼环境中经历低水平的募集。
