Acosta Tomas J, Yoshioka Shin, Komiyama Junichi, Lee Seung-Hyung, Grazul-Bilska Anna T, Skarzynski Dariusz J, Okuda Kiyoshi
Laboratory of Reproductive Endocrinology, Graduate School of Natural Science and Technology, Okayama University, Japan.
J Reprod Dev. 2007 Jun;53(3):473-80. doi: 10.1262/jrd.18142. Epub 2007 Jan 17.
To establish a storage system for isolated bovine luteal endothelial cells (LECs), we investigated the basal and tumor necrosis factor (TNF) alpha-stimulated production of endothelin-1 (ET-1) and prostaglandin (PG) F2alpha in unfrozen and frozen-thawed LECs until passage 10. LECs were obtained from developing corpora lutea (CL; days 5-7 of the estrous cycle) using enzymatic digestion and magnetic beads coated with lectin BS-1. The LECs were frozen at -80 C or further cultured and/or passaged until passage 10 in DMEM/Ham's F-12 supplemented with 10% calf serum. The hormonal productions of unfrozen and frozen/thawed LECs were compared through passages 2-10. When both the unfrozen and frozen/thawed cells reached confluence, the culture medium was replaced with fresh medium containing 0.1% bovine serum albumin (BSA), and the cells were incubated with TNFalpha (50 ng/ml) for 12 h. The basal productions of ET-1 and PGF2alpha by the unfrozen and frozen/thawed LECs were similar at passage 2. The basal production of PGF2alpha by LECs was not altered by passage and storage at -80 C, whereas the basal production of ET-1 decreased from passage 2 and 3 to passage 4 in the unfrozen LECs and from passage 2 to passage 3 in the frozen/thawed LECs. However, production of ET-1 by the unfrozen and frozen/thawed LECs was similar between passages 4-10 and passages 3-10, respectively. Exposure of LECs to TNFalpha increased (P<0.05) ET-1 and PGF2alpha production by the unfrozen and frozen-thawed LECs in all passages examined. Thus, LECs obtained from developing CLs and stored until passage 10 can be used for study of the physiology of LECs in vitro.
为建立分离的牛黄体内皮细胞（LECs）储存系统，我们研究了传代至第10代的未冻存及冻融LECs中内皮素-1（ET-1）和前列腺素（PG）F2α的基础分泌以及肿瘤坏死因子（TNF）α刺激后的分泌情况。使用酶消化法并通过包被凝集素BS-1的磁珠从发育中的黄体（CL；发情周期第5 - 7天）获取LECs。将LECs在-80℃冻存，或进一步培养和/或传代至第10代，培养基为补充有10%小牛血清的DMEM/Ham's F-12。比较传代2 - 10次的未冻存及冻融LECs的激素分泌情况。当未冻存及冻融细胞均达到汇合状态时，将培养基换成含0.1%牛血清白蛋白（BSA）的新鲜培养基，细胞与TNFα（50 ng/ml）孵育12小时。传代2时，未冻存及冻融LECs的ET-1和PGF2α基础分泌相似。LECs的PGF2α基础分泌不受传代及-80℃储存影响，而未冻存LECs中ET-1基础分泌从传代2和3至传代4减少，冻融LECs中从传代2至传代3减少。然而，未冻存及冻融LECs在传代4 - 10次和传代3 - 10次时ET-1分泌分别相似。在所有检测传代中LECs暴露于TNFα均增加（P<0.05）未冻存及冻融LECs的ET-1和PGF2α分泌。因此，从发育中的CL获取并储存至传代10的LECs可用于体外LECs生理学研究。
