Analysis of Cultured Spiral Ligament Fibrocyles for revealing immune mechanisms of the inner ear

1.建立了小鼠螺旋韧带(SL)内三种纤维细胞的免疫细胞化学分类方法。钙调蛋白、S-100蛋白和Na-K-ATPase的联合染色对这种分类是有用的。切取小鼠耳蜗侧壁行二次培养。培养细胞钙调素和S-100蛋白阳性，Na-K-ATPase阴性，提示细胞为I型纤维细胞。用IL-1β或肿瘤坏死因子-α刺激培养细胞，检测各种介质的分泌。刺激后，肿瘤坏死因子-α、IL-6、单核细胞趋化蛋白-1、KC、巨噬细胞趋化蛋白-2、细胞间黏附分子-1和血管内皮生长因子水平升高。这些趋化因子和其他介质的分泌可诱导炎性细胞运动，从而延长炎症反应。用IL-1β或肿瘤坏死因子-α刺激培养细胞，检测各种黏附分子的表达。未经刺激的培养细胞未见VCAM-1、ICAM-1或MAdCAM-1染色。IL-1β刺激后未见表达，而α刺激后可见VCAM1和ICAM-1染色。逆转录聚合酶链式反应显示，α刺激纤维细胞后，VCAM1和ICAM1mRNAs均有表达。这一发现表明，激活的纤维细胞可能导致炎症细胞在SL.5中持续存在。对糖皮质激素受体(GRs)进行了免疫组织化学染色，发现GRs在小鼠内耳各处均有分布。SL是GRs表达较强的地区之一。分别用肿瘤坏死因子-α和地塞米松刺激培养细胞，并与单独用肿瘤坏死因子-α刺激的细胞进行比较。地塞米松抑制成纤维细胞分泌IL-6、MCP-1和KC，提示糖皮质激素对SL成纤维细胞有抗炎作用。

1. An immunocytochemical strategy to classify the three types of fibrocytes found in the murine spiral ligament (SL) was established. A combination of staining for caldesmon, S-100 protein, and Na-K-ATPase was useful for this classification.2. Murine cochlear lateral wall was dissected for secondary culture. Cultures were positive for caldesmon and S-100 protein, and negative for Na-K-ATPase, suggesting that the cells were type I fibrocytes.3. Cultures were stimulated with IL-1β or TNF-α, and secretion of various mediators was measured. After stimulation, TNF-α, IL-6, MCP-1, KC, MIP-2, slCAM-1, and VEGF levels were elevated. Secretion of these chemokines and other mediators could induce inflammatory cell movement, which would prolong the inflammatory response.4. Cultures were stimulated with IL-1β or TNF-α, and expression of various adhesion molecules was investigated. Cultures without stimulation did not show staining for VCAM-1, ICAM-1, or MAdCAM-1. Staining was also absent after stimulation with IL-1β; however, VCAM-1 and ICAM-1 staining was observed after cells were stimulated with TNF-α. RT-PCR analysis showed that both VCAM-1 and ICAM-1 mRNAs were present after fibrocytes were stimulated with TNF-α. The findings suggest that activated fibrocytes may cause inflammatory cells to persist in the SL.5. Immunostaining for glucocorticoid receptors (GRs) was performed, and GRs were found throughout in the murine inner ear. The SL was among the regions where GRs were strongly expressed.6. Secretion of various mediators was measured after cultures were stimulated with TNF-α and dexamethasone, and the results were compared with those of cells stimulated with TNF-α alone. Dexamethasone suppressed secretion of IL-6, MCP-1, and KC by fibrocytes, suggesting that glucocorticoids have an anti-inflammatory effect on SL fibrocytes.