Tang Xinyan, Zhang Caiqiao, Zeng Weidong, Mi Yuling, Liu Hongyun
Department of Veterinary Medicine, College of Animal Sciences, Zhejiang University, No. 268 Kaixuan Road, Hangzhou, Zhejiang 310029, China.
Cell Biol Int. 2006 May;30(5):445-51. doi: 10.1016/j.cellbi.2006.02.004. Epub 2006 Mar 6.
Primordial germ cells (PGCs) are undifferentiated pluripotent stem cells, whose proliferation is influenced by many internal and external factors. In the present study, a PGC-somatic cell co-culture model was established to evaluate effects of the flavonoids daidzein (DAI) and quercetin (QUE) on proliferation of PGCs from embryonic chickens. PGCs were isolated from the germinal ridge of 3.5-4day embryos and cultured in 5% fetal calf serum (FCS)-supplemented Medium 199. PGC subculture was carried out on chicken embryonic fibroblast feeder (CEF) or follicular granulosa cell feeder (GCF) layers. The subcultured PGCs were challenged with flavonoids alone or in combination with a reactive oxygen substance (ROS)-producing system on CEF for 48h. The results showed a better supporting effect of CEF than GCF. Flavonoids (1microg/ml) significantly promoted PGC proliferation, which could be markedly inhibited by ROS. The oxidative damage by ROS was further manifest by decreased superoxide dismutase activity and glutathione levels. In addition, activation of protein kinase A (PKA) by forskolin significantly stimulated PGC proliferation, but PKA inhibitor H89 inhibited the proliferating effects induced by DAI and QUE. These results indicated that cultured PGCs respond to exogenous agents on proliferation and that antioxidant flavonoids could restore the intracellular antioxidant system and promote PGC proliferation via their antioxidant action involving the PKA signaling pathway.
原始生殖细胞（PGCs）是未分化的多能干细胞，其增殖受许多内部和外部因素影响。在本研究中，建立了PGC-体细胞共培养模型，以评估黄酮类化合物大豆苷元（DAI）和槲皮素（QUE）对来自胚胎鸡的PGCs增殖的影响。从3.5 - 4日龄胚胎的生殖嵴中分离出PGCs，并在添加5%胎牛血清（FCS）的199培养基中培养。PGC传代培养在鸡胚成纤维细胞饲养层（CEF）或卵泡颗粒细胞饲养层（GCF）上进行。将传代培养的PGCs单独用黄酮类化合物处理，或与活性氧物质（ROS）产生系统联合在CEF上处理48小时。结果表明，CEF比GCF具有更好的支持效果。黄酮类化合物（1μg/ml）显著促进PGC增殖，而ROS可明显抑制其增殖。ROS导致的氧化损伤进一步表现为超氧化物歧化酶活性降低和谷胱甘肽水平下降。此外，福斯可林激活蛋白激酶A（PKA）显著刺激PGC增殖，但PKA抑制剂H89抑制了DAI和QUE诱导的增殖作用。这些结果表明，培养的PGCs对增殖的外源试剂有反应，抗氧化黄酮类化合物可通过其涉及PKA信号通路的抗氧化作用恢复细胞内抗氧化系统并促进PGC增殖。
