不同培养时间对鸡卵泡颗粒细胞孕酮、雌激素分泌水平及FSHR、LHR基因表达的影响

目的：研究培养不同时间鸡卵泡颗粒细胞孕酮和雌激素的分泌水平，促卵泡素受体（FSHR）和促黄体素受体（LHR）的基因表达水平，推断体外培养时间对颗粒细胞激素分泌及相关受体基因表达的影响。方法：通过细胞体外培养的方法，分别于0 h、24 h、48 h、72 h、96 h收集鸡卵泡颗粒细胞上清液，采用ELISA法测定细胞上清液内的孕酮及雌激素分泌水平，并采用荧光定量PCR技术检测颗粒细胞内FSHR和LHR基因表达情况。结果：在培养初期0 h~48 h孕酮和雌激素分泌量显著降低（P < 0.05），随着培养时间增加到72 h两种激素的分泌量又开始增加，并达到培养初期水平，培养至96 h细胞内孕酮和雌激素分泌量再次降低；颗粒细胞FSHR和LHR mRNA的表达水平则随着培养时间的增加而降低（P < 0.05）。结论：体外培养的卵泡颗粒细胞内孕酮和雌激素的分泌量随体外培养时间的延长呈先降低后升高的趋势，可能与体外培养细胞的生长状态相关，从整体上看随着培养时间的延长，细胞内孕酮和雌激素的分泌量均降低，可能与两种促性腺激素受体FSHR和LHR基因表达量下降相关。

The research of the progesterone and estrogen secretion level and the FSHR,LHR gene quantitative in granular cells cultured in vitro in different time of laying hens

Objective: To research the hormone secretion levels of progesterone and estrogen and the gene expression levels of two go-nadotropin receptors follicle stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) in granular cells of laying hen, and the effect of culture time on the levels of hormone secretion and expression of related receptor gene in granulosa cells was inferred. Methods: The experiment using the method of cells culture in vitro, the granular cells supernatants of hens were collected at 0 h, 24 h, 48 h, 72 h, 96 h, the progesterone and estrogen concentrations in cell supernatants were determined by ELISA kits, and detected the expression of FSHR and LHR gene in granular cells by real-time fluorescent quantitative PCR. Results: The results showed that the progesterone and estrogen secretion reduced in the early culture of 0 h~48 h(P < 0.05), with the culture time increases to 72 h, the secretion of two hormones began to in-crease, and reaching the level of the initial level of culture. When the cells were cultured to 96 h, the rogesterone and estrogen secretion was reduced again. The lower levels of FSHR and LHR mRNA expression in granular cells appeared with the increase of culture time, compared with the group of cell culture to 0 h, the mRNA expression levels of each groups reduced obviously(P < 0.05). Conclusion: The amount of progesterone and estrogen in the cultured follicular granulosa cells decreased with the increase of in vitro culture time, and then increased. This might be related to the growth state of cells cultured in vitro. But on the whole, with the extension of the training time, the secretion of proges-terone and estrogen in the cells decreased. This may be related to the decreased expression of the FSHR and LHR genes in the two go-nadotropin receptors.