PACAP mRNA在大鼠妊娠黄体及离体细胞的表达与调节

目的:观察垂体腺苷酸环化酶激活肽(PACAP)mRNA在大鼠妊娠黄体中的表达及调节。方法:①于妊娠不同时期收集大鼠卵巢。用RT-PCR和原位杂交方法,观察妊娠过程卵巢PACAP mRNA表达的动态变化;②未成年雌性大鼠颈部皮下注射50IU孕马血清促性腺激素,48h后注射25IU人绒毛膜促性腺激素,第6天收集培养黄体细胞。用放免法测定给予不同处理后,培养液中孕酮的含量;用RT-PCR方法检测各组PACAP mRNA表达水平。结果:从妊娠11d起,PACAP mRNA表达逐渐增强,在妊娠19d达高峰;与对照组相比,血小板活化因子(PAF)、福司考林(forskolin)、佛波酯(PMA)均使培养黄体细胞孕酮分泌量及PACAP mRNA表达显著增高(P0.05)。结论:PACAP与中、晚期妊娠的维持密切相关;PAF可促进培养黄体细胞PACAP mRNA的表达,蛋白激酶C(PKC)和蛋白激酶A(PKA)途径都有可能参与了此过程。

Expression and regulation of pituitary adenylate cyclase-activating polypeptide mRNA in pregnant rat corpus luteum

Objective： To investigate the expression changes and regulation of pituitary adenylate cyclase-activating polypeptide（PACAP） mRNA in corpus lutemn during pregnancy. Methods： Pregnant rats＇ ovaries were collected at different time points. The tectmiques of RT-PCR and in situ hybridization were used to observe expression changes of PACAP InRNA in rat ovaries during pregnancy. To further explore the regulation mechanism of PACAP mRNA expression in corpus luteum, luteal cells were cultured in vitro. Immature（25 - 28 days old） female Sprague-Dawley rats were injected subcutaneously with 50IU pregnant mare serum gonadotrophin （PMSG）, and 25IU human chorionic gonadotrophin（hCG）48 h later, to induce folliclular development and luteum formation. On day 6 after hCG administration （the day of hCG administration was the first day）, the rats were killed by guillotine and the ovarian luteal cells were collected. After incubation for 24 h, luteal cells were administration with various factors for 24 h. And then expression changes of PACAP mRNA in luteal cells after administration with different factors were detected by RT-PCR, and radioimmunoassay was used to analyze progesterone levels. Results： With the development of pregnancy, the expression of PACAP mRNA increased gradually, reached the peak at pregnancy 19 d, and then decreased. Compared with control group, platelet activating factor （PAF）, forskolin and PMA could obviously stimulate PACAP mRNA expression in luteal cells which were cultured with corresponding factors for 24 h. At the same time, progesterone levels in culture media were ＇also elevated. Conclusion： PACAP, acting as a local ovary regulator, was closely related to the maintenance of medium-term and late pregnancy. PAF could directly stimulate PACAP mRNA expression in luteal cells, and protein kinase C （PKC） and protein kinase A （PKA） signal pathways could both participate in this process.