Florio P, Vannelli G B, Luisi S, Barni T, Zonefrati R, Falaschi C, Bifulco G, Genazzani A R, Petraglia F
Department of Reproductive Medicine and Child Development, Division of Obstetrics and Gynecology, University of Pisa, Pisa, Italy.
Eur J Endocrinol. 2000 Jul;143(1):133-8. doi: 10.1530/eje.0.1430133.
To evaluate the expression of activin betaA-subunit mRNA and the secretion of activin A in/from cultured GnRH-secreting neuronal cells cloned from human olfactory epithelium (FNC-B4), which showed biochemical and antigenic properties of GnRH-secreting neurons.
FNC-B4 cells were cultured in basal and conditioned media.
Reverse transcription-polymerase chain reaction (RTR-PCR) evaluated the expression of activin betaA-subunit mRNA. By using a specific ELISA, dimeric activin A concentrations were measured in culture media, in the absence or presence of carvone or forskolin and with different doses of progesterone, GnRH, and estradiol.
RT-PCR experiments performed on total RNA isolated from FNC-B4 cells, using specific primers for the activin betaA gene, showed a 787bp DNA band corresponding to the betaA gene. FNC-B4 cells secreted activin A, and the highest accumulation in conditioned medium was achieved after 3h culture: the addition of forskolin, but not of carvone, was able to stimulate the release of activin A from cultured neuronal cells (P<0.01). When progesterone or GnRH was added, a significant accumulation of activin A was observed (P<0.01), while estradiol administration did not significantly affect activin A secretion.
To date, this is the only study, in an in vitro human model reporting, that GnRH-secreting neuronal cells expressed activin betaA-subunit mRNA, and released dimeric activin A in culture medium. The expression and secretion of activin suggests that in these cells activin A might exert its action by autocrine/paracrine mechanisms.
评估从人嗅上皮克隆的分泌促性腺激素释放激素（GnRH）的神经元细胞（FNC - B4）中激活素βA亚基mRNA的表达以及激活素A的分泌情况，该细胞表现出分泌GnRH神经元的生化和抗原特性。
FNC - B4细胞在基础培养基和条件培养基中培养。
逆转录 - 聚合酶链反应（RT - PCR）评估激活素βA亚基mRNA的表达。使用特异性酶联免疫吸附测定（ELISA），在不存在或存在香芹酮或福斯可林以及不同剂量的孕酮、GnRH和雌二醇的情况下，测量培养基中活性二聚体激活素A的浓度。
使用激活素βA基因的特异性引物对从FNC - B4细胞分离的总RNA进行RT - PCR实验，显示出一条对应于βA基因的787bp DNA条带。FNC - B4细胞分泌激活素A，培养3小时后条件培养基中的积累量最高：添加福斯可林而非香芹酮能够刺激培养的神经元细胞释放激活素A（P<0.01）。当添加孕酮或GnRH时，观察到激活素A有显著积累（P<0.01），而给予雌二醇对激活素A的分泌没有显著影响。
迄今为止，这是在体外人类模型报告中唯一一项研究，表明分泌GnRH的神经元细胞表达激活素βA亚基mRNA，并在培养基中释放活性二聚体激活素A。激活素的表达和分泌表明在这些细胞中激活素A可能通过自分泌/旁分泌机制发挥作用。
