Sack Robert A, Sathe Sonal, Beaton Ann, Kozinski Margaret, Bogart Bruce, Lew George, Sharma Savitri, Upponi Anjali
State University of New York State College of Optometry, 33 West 42nd Street at Bryant Park, Manhattan, NY 10036, USA.
Exp Eye Res. 2004 Mar;78(3):371-8. doi: 10.1016/s0014-4835(03)00198-2.
To test whether the cystatin-like functional domain in tear specific lipocalin (TSL) is functionally active in tears during the normal diurnal cycle and during external ocular infections.
Capillary tube collected reflex (RTF), open (OTF) and closed (CTF) eye tear samples were recovered from six normals and semi-quantitatively western blot assayed for cystatin C and TSL. CTF samples were immunoprecipitated with antibodies raised against TSL, cystatin C and other antiproteases and screened for the co-precipitation of proteases by casein and gelatin zymography. OTF samples recovered from individuals with viral, fungal and bacterial keratitis were similarly screened for TSL-bound proteases. Human tissue was subjected to immunohistochemical study.
Western blot analysis reveals a progressive increase in cystatin C in going from RTF to OTF to CTF samples (approximately 3, 7 and 30 ng microl(-1), respectively). In contrast, the concentration of TSL remains constant (approximately 1500 ng microl(-1)). Immunocytochemistry data show staining of the apical surface of the human conjunctiva and some intra-cellular staining for cystatin C, but not for cystatin A. Zymography confirms earlier data that CTF contains exceptionally high levels of proteases bound to a wide range of specific inhibitors. However, only trace amounts of proteases are complexed with cystatin C and no protease can be detected bound to TSL in either the pathological or CTF samples.
Although TSL contains a functional cystatin-like domain, it is not physiologically active during the normal diurnal cycle or during external ocular infections. Reactive proteases in CTF are most likely controlled by the presence of excess levels of more reactive cystatins, especially cystatin C, which accumulates during prolonged eye closure. Immunohistochemical data suggest that the apical conjunctiva may be a contributing source for the accumulating cystatin C.
检测泪液特异性脂钙蛋白（TSL）中类胱抑素功能域在正常昼夜周期及眼表感染期间的泪液中是否具有功能活性。
从6名正常人中收集毛细管采集的反射泪液样本（RTF）、睁眼泪液样本（OTF）和闭眼泪液样本（CTF），采用蛋白质免疫印迹法半定量检测胱抑素C和TSL。用抗TSL、胱抑素C及其他抗蛋白酶的抗体对CTF样本进行免疫沉淀，通过酪蛋白和明胶酶谱法筛选共沉淀的蛋白酶。对从患有病毒性、真菌性和细菌性角膜炎的个体中收集的OTF样本进行类似的TSL结合蛋白酶筛选。对人体组织进行免疫组织化学研究。
蛋白质免疫印迹分析显示，从RTF样本到OTF样本再到CTF样本，胱抑素C呈逐渐增加趋势（分别约为3、7和30 ng/μl）。相比之下，TSL的浓度保持恒定（约1500 ng/μl）。免疫细胞化学数据显示，人结膜上皮表面有胱抑素C染色，细胞内也有一些染色，但胱抑素A无染色。酶谱分析证实了早期的数据，即CTF含有与多种特异性抑制剂结合的异常高水平蛋白酶。然而，只有微量蛋白酶与胱抑素C复合，在病理样本或CTF样本中均未检测到与TSL结合的蛋白酶。
虽然TSL含有一个功能性类胱抑素结构域，但在正常昼夜周期或眼表感染期间它并无生理活性。CTF中的反应性蛋白酶很可能受更具反应性的胱抑素（尤其是胱抑素C）过量存在的控制，胱抑素C在长时间闭眼时会积累。免疫组织化学数据表明，结膜上皮可能是胱抑素C积累的一个来源。
