Ciliary neurotrophic factor released by corneal endothelium surviving oxidative stress ex vivo.
Ciliary neurotrophic factor released by corneal endothelium surviving oxidative stress ex vivo.
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发表时间：
2002-09
影响因子：
4.4
通讯作者：
S. Koh
中科院分区：
文献类型：
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作者：
S. Koh
目的：研究角膜内皮细胞（CE）在角膜氧化应激下释放不具有分泌信号序列的睫状神经营养因子（CNTF）。方法采用Western blot （WB）和免疫沉淀（IP）检测CE细胞和细胞条件培养基（H2O2和PBS置于牛角膜杯中，37℃下培养30分钟）中CNTF和CNTF受体α亚基（cntfr α）的表达。死亡CE细胞的数量由不知道外植体身份的观察者用活力试剂盒在显微镜下测定。在35s蛋氨酸标记（0.1 mCi/mL, 37℃）角膜杯中，CE细胞合成和释放CNTF和cntfr α。结果。新鲜牛眼CE细胞表达25 kda的CNTF，该CNTF可被三种不同的抗体识别。CE细胞表达61 kda的CNTF免疫反应分子（IM），该分子在h2o2条件下的角膜杯中的CE细胞中消失，同时在条件培养基中出现25 kda的CNTF。含有0、0.006、0.012、0.023、0.045、0.09、0.18和0.35 mM H2O2的角膜杯分别显示CE细胞61-kDa CNTF- im的相对水平为100%、84%、77%、61%、52%、39%、35%和35%，而条件培养基中25-kDa CNTF的水平分别为23%、32%、39%、63%、80%、90%、100%和63%。CE细胞表达53 kda的cntfrα，与微量的61 kda cntfrα - im一起出现在条件培养基中，与25 kda CNTF一起出现。H2O2 （0-0.56 mM）不影响CE细胞的活力（每600个细胞中有15个死亡细胞）。在35S-蛋氨酸标记的角膜杯中，CE细胞合成并释放35S 61 kda分子，该分子以h2o2依赖的方式同时具有CNTF-和cntfrα免疫反应，而在（35）s -蛋氨酸标记的培养基中检测到25 kda的CNTF。结论：在亚致死氧化应激下，CE细胞释放自分泌CNTF的机制涉及cntfrα和61 kda CNTF/ cntfrα - im的形成。
PURPOSE To demonstrate that corneal endothelial (CE) cells that survive oxidative stress in corneas release ciliary neurotrophic factor (CNTF), which does not possess the secretion signal sequence. METHODS CNTF and CNTF receptor alpha subunit (CNTFRalpha) in CE cells and cell-conditioned medium (H2O2 and PBS placed in bovine corneal cups for 30 minutes at 37 degrees C) in explant cultures were demonstrated by Western blot (WB) and immunoprecipitation (IP). The number of dead CE cells was determined microscopically with a viability kit, by an observer uninformed of the explants' identities. CNTF and CNTFRalpha synthesis and release by CE cells in 35S-methionine-labeled (0.1 mCi/mL for 8 hours at 37 degrees C) corneal cups were shown by autoradiography and WB. RESULTS. CE cells in fresh bovine eyes expressed a 25-kDa CNTF that was recognized by three different antibodies. CE cells expressed a 61-kDa CNTF-immunoreactive molecule (IM), which disappeared from the CE cells in H2O2-conditioned corneal cups, concomitant with the appearance of the 25-kDa CNTF in the conditioned medium. Corneal cups containing 0, 0.006, 0.012, 0.023, 0.045, 0.09, 0.18, and 0.35 mM H2O2 demonstrated relative levels of CE cell 61-kDa CNTF-IM of 100%, 84%, 77%, 61%, 52%, 39%, 35%, and 35%, respectively, whereas levels of 25-kDa CNTF in the conditioned medium were 23%, 32%, 39%, 63%, 80%, 90%, 100%, and 63%, respectively. CE cells expressed a 53-kDa CNTFRalpha that, along with trace amounts of a 61-kDa CNTFRalpha-IM, appeared concomitantly with the 25-kDa CNTF in the conditioned medium. H2O2 (0-0.56 mM) did not affect the viability of CE cells (15 dead cells per 600 cells). CE cells in 35S-methionine-labeled corneal cups synthesized and released a 35S 61-kDa molecule that was both CNTF- and CNTFRalpha-immunoreactive in an H2O2-dependent manner, whereas 25-kDa CNTF was detected in the (35)S-methionine labeling medium. CONCLUSIONS CE cells release autocrine CNTF under sublethal oxidative stress by a mechanism that involves CNTFRalpha and the formation of a 61-kDa CNTF/CNTFRalpha-IM.