MUCINS OF THE OCULAR SURFACE

泪膜的粘液负责维持眼睛表面的液体，并提供微生物屏障以保护眼睛免受感染。眼表疾病，如干眼症、维生素A缺乏症、眼表感染以及过敏性结膜炎，可能涉及黏液缺乏、黏液层破坏或大量黏液排出。在之前的资助期间，我们证明了三个粘蛋白基因在眼表上皮中表达，其中两个普遍的是跨膜粘蛋白MUC4和杯状细胞特异性粘蛋白MUC5AC。我们对MUC4的部分进行了测序，并为这两种粘蛋白开发了探针、抗体和检测方法，我们现在建议在四个特定的目标中使用，以了解正常和病理状态下这些粘蛋白在眼表上的功能和表达调控。目的一：描述眼表跨膜黏液蛋白MUC4的两个方面。A.确定粘蛋白是否仍然与顶膜糖萼相关，或者其细胞外结构域是否脱落到泪膜中。b.测试候选MUC4基因表达诱导剂，基于从MUC4调控区测序中鉴定出的推定转录因子结合位点的存在，以及表明其在MUC4调控中的潜在作用的初步数据。目的二：我们假设结膜杯状细胞分化的特征是诱导MUC5AC基因的表达，并且这种诱导可以受到环境刺激和细胞效应物的调节。我们建议：a.在小鼠模型中确定杯状细胞分化/Muc5AC表达是否会受到表面刺激、感染或特定过敏原的影响；b.根据MUC5AC启动子区存在的调控元件和已知影响胃肠道杯状细胞分化的培养条件，确定结膜杯状细胞分化是否可以在体外增强。目的III：确定MUC5AC是否对MUC4和泪膜、溶菌酶和分泌性IgA中普遍存在的杀菌蛋白具有特异性亲和力。目的IV：确定特定类型的干眼（干燥综合征）和季节性变应性结膜炎中MUC4和MUC5AC mRNA和蛋白的含量是否与正常人群不同。我们假设，干眼综合征的特征是由于MUC5AC和MUC4蛋白的减少而导致表面湿润的丧失，而过敏性结膜炎的特征是粘蛋白的增加，以促进过敏原的去除。

The mucus of the tear film is responsible for maintenance of fluid on the surface of the eye and for providing a microbe barrier to protect the eye from infection. Ocular surface diseases such as those of the dry eye type, vitamin A deficiency, ocular surface infections as well as allergic conjunctivits may involve mucus deficiency, disruption of the mucus layer, or discharge of large amounts of mucus. During the previous funding period, we demonstrated that three mucin genes are expressed by the ocular surface epithelium, two prevalent ones being the membrane- spanning mucin MUC4 and the goblet cell-specific mucin MUC5AC. We sequenced portions of MUC4 and developed probes, antibodies, and assay methods for both mucins that we now propose to use in four specific aims toward understanding aspects of the function and regulation of expression of these mucins on the ocular surface in normal and pathologic states. Aim I: Characterize two aspects of the membrane-spanning mucin MUC4 on the ocular surface. A. determine whether the mucin remains associated with the apical membrane glycocalyx or whether its extracellular domain is shed into the tear film. b. Test candidate inducers of MUC4 gene expression, based on presence of putative transcription factor binding sites identified from sequencing the MUC4 regulatory region and on preliminary data indicating their potential role in its regulation. Aim II: We hypothesize that conjunctival goblet cell differentiation is characterized by induction of expression of the MUC5AC gene and that such induction can be regulated by environmental stimuli as well as cellular effectors. We propose to: a. determine in a mouse model whether goblet cell differentiation/Muc5AC expression can be influenced by surface irritants, infections, or specific allergens; b. determine whether conjunctival goblet cell differentiation can be enhanced in vitro, based on demonstrated presence of regulatory elements in the promoter region of MUC5AC and on culture conditions known to affect gastrointestinal goblet cell differentiation. Aim III: Determine if MUC5AC has specific affinities for MUC4 and the bactericidal proteins prevalent in the tear film, lysozyme, and secretory IgA. Aim IV: Determine in a specific type of dry eye (Sjogren's syndrome), and in seasonal allergic conjunctivitis whether amounts of MUC4 and MUC5AC mRNA and protein differ from the normal population. We hypothesize that dry eye syndromes are characterized by loss of surface wetting due to reduced amounts of MUC5AC and MUC4 protein, whereas, allergic conjunctivitis is characterized by an increased amount of mucins to facilitate allergen removal.