Expression data from Spdef +/+ and Spdef -/- mice

Goblet cell numbers decrease within the conjunctival epithelium in drying and cicatrizing ocular surface diseases. Factors regulating goblet cell differentiation in conjunctival epithelium are unknown. Recent data indicate that the transcription factor SAM-pointed domain epithelial-specific transcription factor (Spdef) is essential for goblet cell differentiation in tracheobronchial and gastrointestinal epithelium of mice. Using Spdef -/- mice, we determined that Spdef is required for conjunctival goblet cell differentiation and that Spdef -/- mice, which lack conjunctival goblet cells, have significantly increased corneal surface fluorescein staining and tear volume, a phenotype consistent with dry eye. Microarray analysis of conjunctival epithelium in Spdef -/- mice identified 43 signficantly upregulated genes and 110 signficantly downregulated genes in the conjunctival epithelium of Spdef -/- mice compared to that of Spdef +/+ control mice (3 fold change, p<0.01). Downregulated genes of particular interested included goblet cell-specific genes (Muc5ac, Tff1, Gcnt3). Upregulated genes included epithelial cell differentiation/keratinization genes (Sprr2h, Tgm1) and pro-inflammatory genes (Il1-alpha, Il-1beta, Tnf-apha), all of which are upregulated in dry eye. Interestingly, four Wnt pathway genes were downregulated. Conjunctival epithelium of Spdef +/+ and Spdef -/- mice was collected by laser capture microdissection for RNA extraction and hybridization on Affymetrix microarrays to determine if gene expression patterns in the conjunctival epithelium of Spdef -/- mice is altered compared to that of Spdef +/+ mice.在干燥性与瘢痕化性眼表疾病中，结膜上皮内的杯状细胞数目会出现减少。目前，调控结膜上皮杯状细胞分化的相关因子仍未明确。近期研究表明，转录因子SAM-pointed结构域上皮特异性转录因子（SAM-pointed domain epithelial-specific transcription factor，Spdef）对小鼠气管支气管上皮与胃肠道上皮的杯状细胞分化具有不可或缺的作用。本研究利用Spdef -/-小鼠开展实验，证实Spdef是结膜杯状细胞分化所必需的因子；缺失结膜杯状细胞的Spdef -/-小鼠，其角膜荧光素染色评分与泪液体积均显著升高，表型与干眼症相符。对Spdef -/-小鼠的结膜上皮进行基因微阵列分析后发现，与Spdef +/+野生型对照小鼠相比，Spdef -/-小鼠的结膜上皮中有43个基因显著上调、110个基因显著下调（差异倍数≥3倍，p<0.01）。其中值得关注的下调基因包括杯状细胞特异性基因（Muc5ac、Tff1、Gcnt3）。上调基因则涵盖上皮细胞分化/角化相关基因（Sprr2h、Tgm1）与促炎基因（Il1-α、Il-1β、Tnf-α），上述基因在干眼症中均呈现上调表达。有趣的是，有4个Wnt信号通路基因出现下调表达。本研究通过激光捕获显微切割技术采集Spdef +/+与Spdef -/-小鼠的结膜上皮，提取RNA后在Affymetrix基因芯片上进行杂交，以对比分析两组小鼠结膜上皮的基因表达模式是否存在差异。