MUCINS OF THE OCULAR SURFACE

泪膜的粘液负责维持眼睛表面的液体，并提供一种微生物屏障来保护眼睛免受感染。眼表疾病，如干眼型、维生素A缺乏、眼表感染以及过敏性结膜炎，可能涉及粘液缺乏、粘液层破坏或大量粘液排出。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细胞中表达，其中两个流行的基因是跨膜粘蛋白MUC4和杯状细胞特异性粘蛋白MUC5AC。我们对MUC4的部分序列进行了测序，并开发了针对这两种粘蛋白的探针、抗体和检测方法，我们现在建议将其用于四个特定的目的，以了解这些粘蛋白在正常和病理状态下的功能和表达调节。目的I：对眼表面跨膜粘蛋白MUC4的两个方面进行表征。确定粘蛋白是否仍然与顶膜糖基化有关，或者它的胞外区域是否脱落到泪膜中。B.测试MUC4基因表达的候选诱导物，基于从MUC4调节区测序中确定的假定转录因子结合位点的存在以及表明它们在其调控中的潜在作用的初步数据。目的II：我们假设结膜杯状细胞分化的特征是诱导MUC5AC基因的表达，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的表达是否可以受到表面刺激物、感染或特定过敏原的影响；B.基于MUC5AC启动子区域存在调节元件和已知影响胃肠道杯状细胞分化的培养条件，确定体外是否可以促进结膜杯状细胞分化。目的III：确定MUC5AC是否与MUC4以及泪膜、溶菌酶和分泌型IgA中普遍存在的杀菌蛋白具有特异性亲和力。目的：检测特定类型干眼(干燥综合征)和季节性过敏性结膜炎中MUC4和MUC5AC的mRNA和蛋白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增加以促进过敏原的清除。

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.