Department of Ophthalmology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Korea.
Invest Ophthalmol Vis Sci. 2014 Jan 29;55(1):535-41. doi: 10.1167/iovs.13-12459.
To evaluate the effects of nerve growth factor (NGF), which is activated during inflammatory episodes of ocular diseases, on the apoptotic response in cultured human primary conjunctival epithelial cells (pHCECs).
Levels of NGF transcripts and NGF protein in pHCEC grown in medium with normal osmolarity (307 mOsm/L) or hyperosmolar medium (350, 400, and 450 mOsm/L) were determined using RT-PCR or ELISA, respectively. To assess apoptosis, pHCEC were cultured in normal or 400 mOsm/L hyperosmolar medium with neutralizing anti-NGF antibody or recombinant human NGF for 6 hours before analysis by flow cytometry. Levels of Bcl-xL, Bax, phosphorylated JNK, and cleaved caspase-3 were detected using Western blotting. Levels of the inflammatory cytokine IL-6 was analyzed using ELISA.
Hyperosmolar conditions increased NGF levels in cultured pHCECs. Hyperosmolarity and exposure to neutralizing anti-NGF antibody significantly increased the number of apoptotic cells. Addition of recombinant human NGF to 400 mOsm/L medium decreased the number of apoptotic cells, reduced the expression of phosphorylated JNK, Bax, and cleaved caspase-3 and increased the expression of Bcl-xL. Levels of IL-6 were increased by hyperosmotic stress but decreased by exposure to recombinant human NGF.
Hyperosmolarity induces apoptosis of pHCECs by activating JNK signaling. Increased levels of NGF under hyperosmolar conditions may contribute, at least in part, to the reduced apoptosis of pHCECs and may be beneficial in recovering conjunctival damage due to chronic hyperosmolar stress.
评估神经生长因子（NGF）在眼部疾病炎症发作期间被激活后对培养的人原代结膜上皮细胞（pHCEC）凋亡反应的影响。
采用 RT-PCR 或 ELISA 分别测定在渗透压正常（307 mOsm/L）或高渗培养基（350、400 和 450 mOsm/L）中培养的 pHCEC 中 NGF 转录本和 NGF 蛋白的水平。为了评估凋亡，将 pHCEC 在正常或 400 mOsm/L 高渗培养基中培养 6 小时，然后用流式细胞术进行分析，同时培养基中加入中和抗 NGF 抗体或重组人 NGF。采用 Western blot 检测 Bcl-xL、Bax、磷酸化 JNK 和 cleaved caspase-3 的水平。采用 ELISA 分析炎症细胞因子 IL-6 的水平。
高渗条件增加了培养的 pHCEC 中的 NGF 水平。高渗和暴露于中和抗 NGF 抗体显著增加了凋亡细胞的数量。在 400 mOsm/L 培养基中加入重组人 NGF 可减少凋亡细胞的数量，降低磷酸化 JNK、Bax 和 cleaved caspase-3 的表达，并增加 Bcl-xL 的表达。高渗应激增加了 IL-6 的水平，但降低了重组人 NGF 的表达。
高渗通过激活 JNK 信号诱导 pHCEC 凋亡。高渗条件下 NGF 水平的升高可能至少部分导致 pHCEC 凋亡减少，并可能有益于恢复由于慢性高渗应激引起的结膜损伤。
