Murakami Shuko, Shibaya Masami, Takeuchi Kosuke, Skarzynski Dariusz J, Okuda Kiyoshi
Laboratory of Reproductive Endocrinology, Faculty of Agriculture, Okayama University, Japan.
J Reprod Dev. 2003 Dec;49(6):531-8. doi: 10.1262/jrd.49.531.
To establish a storage system for isolated endometrial cells, we investigated the basal, oxytocin (OT)- and tumor necrosis factor (TNF) alpha-stimulated production of prostaglandin (PG) F(2alpha) in bovine-passaged and frozen-thawed endometrial cells. Stromal and epithelial cells obtained from cows in the early stage of the estrous cycle (Days 2-5) were frozen at -80 C or further cultured and/or passaged until passage 4 in DMEM/Ham's F-12 supplemented with 10% calf serum. A fresh-unfrozen primary culture and one-time passaged fresh-unfrozen cells were used as the control. When both unfrozen and frozen cells reached confluence, the culture medium was replaced with fresh medium with 0.1% BSA and the cells were stimulated with OT (100 ng/ml) or TNFalpha (1 ng/ml) for 4 h. The passage and freezing of the endometrial cells did not affect their morphology. In primary culture of frozen and unfrozen endometrial cells, OT strongly stimulated PGF(2alpha) production in epithelial cells, and TNFalpha strongly stimulated PGF(2alpha) production in stromal cells (P<0.05). The basal output of PGF(2alpha) in frozen stromal cells was similar to that in unfrozen stromal cells. However, the basal output of PGF(2alpha) in frozen epithelial cells was significantly lower than that unfrozen cells (P<0.05). On the other hand, in passaged cells, the basal level of PGF(2alpha) production was retained until passage 1 in epithelial cells, whereas it was retained until passage 4 in stromal cells. Although epithelial cells responded to OT in PGF(2alpha) production until passage 2 (P<0.05), the stromal cells showed a significant response to TNFalpha until passage 4 (P<0.05). These results suggest that stored cells could be used for studying the physiology of bovine endometrium in vitro until passage 1 in endometrial epithelial cells, and until passage 4 in stromal cells.
为建立分离的子宫内膜细胞储存系统，我们研究了基础状态下以及催产素（OT）和肿瘤坏死因子（TNF）α刺激后，传代培养及冻融处理的牛子宫内膜细胞中前列腺素（PG）F2α的产生情况。从发情周期早期（第2 - 5天）的奶牛获取的基质细胞和上皮细胞，在补充有10%小牛血清的DMEM/Ham's F - 12培养基中，于-80°C冷冻，或进一步培养和/或传代至第4代。以新鲜未冷冻的原代培养细胞和一次性传代的新鲜未冷冻细胞作为对照。当未冷冻和冷冻的细胞均达到汇合状态时，将培养基更换为含0.1%牛血清白蛋白的新鲜培养基，并用OT（100 ng/ml）或TNFα（1 ng/ml）刺激细胞4小时。子宫内膜细胞的传代和冷冻并未影响其形态。在冷冻和未冷冻的子宫内膜细胞原代培养中，OT强烈刺激上皮细胞中PGF2α的产生，而TNFα强烈刺激基质细胞中PGF2α的产生（P<0.05）。冷冻基质细胞中PGF2α的基础产量与未冷冻基质细胞相似。然而，冷冻上皮细胞中PGF2α的基础产量显著低于未冷冻细胞（P<0.05）。另一方面，在传代细胞中，上皮细胞中PGF2α产生的基础水平在传代至第2代时保持不变（P<0.05），而基质细胞中则保持至传代第4代。尽管上皮细胞在传代至第2代时对OT刺激PGF2α产生仍有反应（P<0.05），但基质细胞对TNFα的显著反应可持续至传代第4代（P<0.05）。这些结果表明，储存的细胞可用于体外研究牛子宫内膜的生理学，其中子宫内膜上皮细胞可使用至传代第1代，基质细胞可使用至传代第4代。
