权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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

分析培养的螺旋韧带纤维环以揭示内耳的免疫机制

基本信息

批准号：
11671689
负责人：
ICHIMIYA Issei
金额：
$ 2.24万
依托单位：
Oita Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2002
项目状态：
已结题

项目摘要

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.

1.建立了一种免疫细胞化学策略来对小鼠螺旋韧带（SL）中发现的三种类型的纤维细胞进行分类。 caldesmon、S-100 蛋白和 Na-K-ATP 酶染色的组合对于这种分类很有用。2．解剖小鼠耳蜗侧壁进行二次培养。培养物中caldesmon和S-100蛋白呈阳性，Na-K-ATPase呈阴性，提示细胞为I型纤维细胞。 3．用 IL-1β 或 TNF-α 刺激培养物，并测量各种介质的分泌。刺激后，TNF-α、IL-6、MCP-1、KC、MIP-2、slCAM-1 和 VEGF 水平升高。这些趋化因子和其他介质的分泌可诱导炎症细胞运动，从而延长炎症反应。4．用 IL-1β 或 TNF-α 刺激培养物，并研究各种粘附分子的表达。未经刺激的培养物未显示 VCAM-1、ICAM-1 或 MAdCAM-1 染色。用 IL-1β 刺激后也没有染色；然而，用TNF-α刺激细胞后观察到VCAM-1和ICAM-1染色。 RT-PCR 分析显示，用 TNF-α 刺激纤维细胞后，VCAM-1 和 ICAM-1 mRNA 均存在。研究结果表明，活化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 (GR) 进行免疫染色，在小鼠内耳中发现了 GR。 SL是GRs强表达的区域之一。6．用TNF-α和地塞米松刺激培养物后测量各种介质的分泌，并将结果与单独用TNF-α刺激的细胞的结果进行比较。地塞米松抑制纤维细胞分泌 IL-6、MCP-1 和 KC，表明糖皮质激素对 SL 纤维细胞具有抗炎作用。