Mast cell chymase induces conjunctival epithelial cell apoptosis by a mechanism involving degradation of fibronectin
Mast cell chymase induces conjunctival epithelial cell apoptosis by a mechanism involving degradation of fibronectin
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DOI：
10.1080/02713680590959367
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发表时间：
2005-06-01
影响因子：
2
通讯作者：
Murakami, A
中科院分区：
文献类型：
--
作者：
Ebihara, N;Takai, S;Murakami, A
目的：为探讨春季角结膜炎结膜上皮损伤的机制，我们研究了人糜酶对体外培养的结膜上皮细胞的作用。研究方法：将人结膜上皮细胞与人糜酶一起孵育24或48小时，其活性水平可能存在于春季角结膜炎患者的泪液中。相差显微镜观察细胞形态学变化。为了确定脱落细胞的数量，我们使用自动细胞计数器，而凋亡细胞通过流式细胞术定量。ELISA法测定条件培养液中可溶性纤维连接蛋白的含量。结果如下：大多数细胞在与糜蛋白酶孵育24小时后脱落。然而，糜蛋白酶介导的凋亡是一个缓慢的过程，只有在与糜蛋白酶孵育36至48小时后才能检测到。当细胞在纤连蛋白包被的平板中孵育时，细胞脱落和凋亡都被阻断。可溶性纤维连接蛋白的增加依赖于糜酶的加入量和暴露时间。半胱天冬酶抑制剂（抗凋亡剂）拯救细胞免于凋亡，但不能防止细胞脱离。这些结果表明，凝乳酶诱导的结膜上皮细胞凋亡代表失巢凋亡，这是一个缓慢发生的凋亡过程中诱导缺乏粘附到细胞外基质。结论：人肥大细胞糜酶通过降解纤维连接蛋白引起结膜上皮细胞脱落，导致继发性凋亡。
Purpose: To determine the mechanism of conjunctival epithelial injury in vernal keratoconjunctivitis, we investigated the effects of human chymase on conjunctival epithelial cells in vitro. Methods: Human conjunctival epithelial cells were incubated with human chymase for 24 or 48 hr at levels of activity that were likely to exist in the tear fluid of patients with vernal keratoconjunctivitis. Morphologic changes of the cells were observed by phase contrast microscopy. To determine the number of detached cells, we used an automated cell counter, while apoptotic cells were quantitated by flow cytometry. The level of soluble fibronectin in conditioned medium was measured by ELISA. Results: Most of the cells in the incubation with chymase were detached by 24 hr. However, chymase-mediated apoptosis was a slower process and was only detected after incubation of cells with chymase for 36 to 48 hr. Both cell detachment and apoptosis were blocked when cells were incubated in fibronectin-coated plates. The increase of soluble fibronectin was dependent on the amount of chymase added and the exposure time. A caspase inhibitor (antiapoptotic agent) rescued cells from apoptosis but did not prevent cell detachment. These results indicate that chymase-induced apoptosis of conjunctival epithelial cells represents anoikis, which is a slowly occurring apoptotic process induced by lack of adhesion to an extracellular matrix. Conclusions: Human mast cell chymase caused conjunctival epithelial cell detachment by degrading fibronectin, and this led to secondary apoptosis.