Synergistic effect of platelet-activating factor and tumor necrosis factor-α on corneal myofibroblast apoptosis
Synergistic effect of platelet-activating factor and tumor necrosis factor-α on corneal myofibroblast apoptosis
复制标题
DOI：
10.1167/iovs.05-0581
复制
发表时间：
2006-03-01
影响因子：
4.4
通讯作者：
Bazan, HEP
中科院分区：
文献类型：
--
作者：
He, JC;Bazan, HEP
目的。角膜损伤修复后肌成纤维细胞的消除对于维持角膜透明至关重要，本研究探讨血小板活化因子(PAF)联合肿瘤坏死因子(TNF)-α在角膜肌成纤维细胞凋亡中的作用。猪角膜肌成纤维细胞 (PCM) 是从以低密度 (5 个细胞/mm(2)) 铺板的传代培养成纤维细胞中获得的。使用小鼠抗平滑肌肌动蛋白抗体来鉴定细胞表型。进行免疫荧光来定位这些细胞中的 PAF 和 TNF-α 受体。通过蛋白质印迹分析来表征抗体的反应性。为了诱导肌成纤维细胞凋亡，用甲基氨甲酰-PAF（cPAF，300 nM）（一种不可水解的 PAF 类似物）、TNF-α（20 ng/mL）和 TNF-α+cPAF（有或没有 LAU-0901（150 nM）（一种新型 PAF 拮抗剂））处理 PCM 24 至 72 小时。通过 Hoechst 33258 和 TUNEL 染色以及 DNA 梯式分析检测细胞凋亡。 6-二脒基-2-苯基吲哚 (DAPI) 用于核复染。通过荧光显微镜记录图像。结果。 PAF 受体（N 末端）多克隆抗体的免疫荧光显示该受体在肌成纤维细胞的质膜和核膜中表达。 TNF-α 受体 II (TNF-RII) 位于细胞质中，而 TNF-受体 I (TNF-RI) 则存在于细胞质和质膜中。用TNF-α处理24、48和72小时分别诱导18%、24%和32%的肌成纤维细胞凋亡。蛋白质印迹分析显示与受体分子量相对应的单条带的表达。 cPAF 处理分别诱导 10%、18% 和 26% 的细胞凋亡。然而，两种细胞因子的处理分别诱导了 42%、78% 和 86% 的细胞凋亡，证明了 PAF 和 TNF-a 之间的协同作用。用 LAU-0901 阻断 PAF 受体可抑制 PAF 诱导的协同效应。结论。角膜肌成纤维细胞在核膜中表达PAF受体，并且还表达TNF-RI和RII。 PAF 和 TNF-a 对肌成纤维细胞凋亡的协同作用表明，在角膜基质伤口愈合过程中，PAF 与其他细胞因子的联合作用在消除这些细胞方面发挥着重要作用。
PURPOSE. Elimination of myofibroblasts after repair of corneal injury is essential for the maintenance of corneal transparenc, In the current study, the role of platelet-activating factor (PAF) in combination with tumor necrosis factor (TNF)-alpha in corneal myofibroblast apoptosis was explored.METHODS. Porcine corneal myofibroblasts (PCMs) were obtained from subcultured fibroblasts plated at a low density (5 cellS/mm(2)). Mouse anti-a-smooth muscle actin antibody was used to identify the cell phenotype. Immunofluorescence was performed to localize PAF and TNF-alpha receptors in those cells. The reactivity of the antibodies was characterized by Western blot analysis. To induce myofibroblast apoptosis, PCMs were treated for 24 to 72 hours with methylcarbamyl-PAF (cPAF, 300 nM), a nonhydrolyzable PAF analogue, TNF-alpha (20 ng/mL), and TNF-alpha+cPAF, with or without LAU-0901 (150 nM), a novel PAF antagonist. Apoptosis was assayed by Hoechst 33258 and TUNEL staining and DNA laddering. 6-Diamidino-2-phenylindole (DAPI) was used for nuclear counterstaining. Images were recorded by fluorescence microscope.RESULTS. Immunofluorescence with a PAF-receptor (N terminus) polyclonal antibody showed that the receptor was expressed in both plasma and nuclear membranes of myofibroblasts. TNF-alpha receptor II (TNF-RII) was localized in the cytoplasm, whereas TNF-receptor I (TNF-RI) was found in both cytoplasm and plasma membrane. Treatment with TNF-a for 24, 48, and 72 hours induced apoptosis in 18%, 24%, and 32%, respectively, of the myofibroblasts. Western blot analysis showed expression of single bands corresponding to the molecular weights of the receptors. Treatment with cPAF induced apoptosis in 10%, 18%, and 26% of the cells, respectively. However, treatment with both cytokines induced apoptosis in 42%, 78%, and 86%, respectively, of the cells, demonstrating a synergistic action between PAF and TNF-a. Blocking the PAF receptor with LAU-0901 inhibited the synergistic effect induced by PAF.CONCLUSIONS. Corneal myofibroblasts express a PAF receptor in the nuclear membrane, and they also express TNF-RI and RII. The synergistic effect on myofibroblast apoptosis by PAF and TNF-a suggests that during corneal stromal wound healing, PAF acting in conjunction with other cytokines could play an important role in eliminating these cells.