Haque M S, Arora J K, Dikdan G, Lysz T W, Zelenka P S
Laboratory of Molecular and Developmental Biology, National Eye Institute, Bethesda, MD 20892, USA.
Mol Vis. 1999 Jun 15;5:8.
Cultured rat lenses and primary human lens epithelial cells (HLECs) express12-lipoxygenase (12-LOX) and require a 12-LOX metabolite of arachidonic acid for growth in response to EGF and insulin. This study seeks to identify an established cell line with these characteristics.
Immunoblotting was used to screen eight lens epithelial cell lines for 12-LOX expression: the human line, HLE-B3; mouse lines alphaTN4, 17EM15, 21EM15, and MLE6, and rabbit lines N/N1003A, LEP2 and B3. DNA synthesis was measured as incorporation of 3H-thymidine into DNA. Expression of c-fos mRNA was detected by RT-PCR. The involvement of 12-lipoxygenase metabolites was determined using the lipoxygenase inhibitors baicalein, cinnamyl 3,4-dihydroxy-alpha-cyanocinnamate (CDC), or nordihydroguiairetic acid (NDGA).
12-LOX was detected only in the rabbit lines N/N1003A, LEP2 and B3. N/N1003A cells were chosen for further study. 12-LOX inhibitors blocked DNA synthesis in response to EGF with or without insulin. Inhibition of EGF-stimulated DNA synthesis was reversed by 0.3 microM to 3 microM 12(S)hydroxyeicosatetraenoic acid (HETE), but not by equivalent concentrations of 5(S)HETE, 8(S)HETE, 15(S)HETE, or 12(R)HETE. Baicalein prevented EGF induction of c-fos mRNA. The transformed HLEC line, HLE-B3, showed little stimulation of DNA synthesis in response to EGF and was unaffected by the presence of 12-LOX inhibitors.
N/N1003A cells, like primary cultured human lens epithelial cells or neonatal rat lenses, require 12-LOX activity for EGF dependent growth. This line will be useful for studies of the mechanism of action of 12(S)HETE.
培养的大鼠晶状体和原代人晶状体上皮细胞（HLECs）表达12-脂氧合酶（12-LOX），并且需要花生四烯酸的一种12-LOX代谢产物以响应表皮生长因子（EGF）和胰岛素来实现生长。本研究旨在鉴定一种具有这些特征的成熟细胞系。
采用免疫印迹法筛选8种晶状体上皮细胞系的12-LOX表达情况，包括人源细胞系HLE-B3；小鼠细胞系alphaTN4、17EM15、21EM15和MLE6，以及兔细胞系N/N1003A、LEP2和B3。通过测量3H-胸腺嘧啶核苷掺入DNA的情况来检测DNA合成。采用逆转录聚合酶链反应（RT-PCR）检测c-fos mRNA的表达。使用脂氧合酶抑制剂黄芩素、肉桂酰3,4-二羟基-α-氰基肉桂酸酯（CDC）或去甲二氢愈创木酸（NDGA）来确定12-脂氧合酶代谢产物的参与情况。
仅在兔细胞系N/N1003A、LEP2和B3中检测到12-LOX。选择N/N1003A细胞进行进一步研究。12-LOX抑制剂可阻断在有或无胰岛素情况下对EGF作出反应的DNA合成。0.3微摩尔至3微摩尔的12(S)-羟基二十碳四烯酸（HETE）可逆转对EGF刺激的DNA合成的抑制作用，但同等浓度的5(S)-HETE、8(S)-HETE、15(S)-HETE或12(R)-HETE则不能。黄芩素可阻止EGF诱导c-fos mRNA。转化的HLEC细胞系HLE-B3对EGF刺激的DNA合成几乎没有反应，并且不受12-LOX抑制剂存在的影响。
N/N1003A细胞与原代培养的人晶状体上皮细胞或新生大鼠晶状体一样，依赖12-LOX活性实现依赖EGF的生长。该细胞系将有助于研究12(S)-HETE的作用机制。
