Saso L, Tommasino P, Grippa E, Leone M G, Silvestrini B
Department of Pharmacology of Natural Substances and General Physiology, University of Rome La Sapienza, Italy.
Res Commun Mol Pathol Pharmacol. 1999 Feb;103(2):149-65.
Although it is well established that ocular mucins and other proteins are essential for tear film stability, whether certain drugs, like non steroidal antiinflammatory drugs (NSAIDs), could cause ocular dryness by inhibiting their secretion is not known. To perform these and other studies of pharmacological interest, we evaluated several micromethods for the analysis of tear samples. The major proteins of the tear fluid collected in capillaries, i.e. IgA, lactoferrin, tear specific prealbumin and lysozyme, were analyzed by SDS-PAGE and gel permeation HPLC, using 2.5-5 microliters of sample. Gastric mucin (PGM), examined as a standard, was analyzed by solid phase assays based on previously described histochemical staining methods: dot blot assays were performed using small disks of polyvinylidene difluoride or nylon membranes, prepared by an ordinary paper punch, which were coated with PGM and stained by Alcian blue or the periodic acid Schiff's reagent. The densitometric analysis was carried out using an ordinary flat scanner controlled by a personal computer equipped with an inexpensive software. The sensitivity of these simple assays was low (100-500 micrograms) but considered sufficient for certain studies. A more sensitive assay (5-20 micrograms) was developed by immobilizing PGM in small agarose gels (100 microliters), prepared in the wells of 96-well microplates, which could by stained by stains-all and analyzed by an automatic plate reader at 595 nm.
虽然眼黏蛋白和其他蛋白质对泪膜稳定性至关重要已得到充分证实，但某些药物，如非甾体抗炎药（NSAIDs），是否会通过抑制其分泌而导致眼干尚不清楚。为了进行这些及其他具有药理学意义的研究，我们评估了几种分析泪液样本的微量方法。使用2.5 - 5微升样本，通过SDS - PAGE和凝胶渗透HPLC分析收集在毛细管中的泪液主要蛋白质，即IgA、乳铁蛋白、泪液特异性前白蛋白和溶菌酶。作为标准进行检测的胃黏蛋白（PGM），通过基于先前描述的组织化学染色方法的固相测定进行分析：使用由普通打孔器制备的聚偏二氟乙烯或尼龙膜小圆盘进行斑点印迹测定，这些小圆盘用PGM包被并用阿尔辛蓝或过碘酸希夫试剂染色。使用配备廉价软件的个人计算机控制的普通平板扫描仪进行光密度分析。这些简单测定的灵敏度较低（100 - 500微克），但认为对某些研究而言足够了。通过将PGM固定在96孔微孔板孔中制备的小琼脂糖凝胶（100微升）中开发了一种更灵敏的测定方法（5 - 20微克），该凝胶可用通用染色剂染色并通过自动酶标仪在595 nm处进行分析。
