补肾调经方含药血清对体外培养的人卵巢颗粒细胞FSH/cAMP-PKA通路的影响

目的通过补肾调经方含药血清对体外培养的人卵巢颗粒细胞卵泡刺激素／环磷酸腺苷-蛋白激酶A（follicle stimulating hormone／cyclic adenosine monophosphate-protein kinaseA，FSH／cAMP-PKA）通路的影响，探讨补肾调经方改善卵巢内分泌功能可能的分子机制。方法收集行体外受精一胚胎移植（In vitro fertilization-embryo transfer，IVF．ET）患者卵泡液，提取颗粒细胞进行原代细胞培养24h，以低、中、高剂量补肾调经方人、大鼠含药血清以及正常人、大鼠血清分别与体外培养的人卵巢颗粒细胞共同孵育48h，分为补肾调经方低、中、高剂量组以及对照组。采用放射免疫方法测定培养液中雌二醇（estrogen，E2）、孕酮（progesterone，P）、环磷酸腺苷（cyclic adenosine monophosphate，cAMP）的水平，采用蛋白印迹法和Real-timePCR分别检测卵泡刺激素受体（follicle stimulating hormone receptor，FSHR）蛋白和FSHR、P450芳香化酶（P450 aromatase，P450arom）mRNA在颗粒细胞的表达。结果人含药血清组：与对照组比较，补肾调经方中、高剂量组培养液中E2、cAMP水平均明显升高（P〈0．05），补肾强经方中剂量纽P明显降低（P〈0．01）；补肾调经方中、高剂量组卵巢颗粒细胞FSHR蛋白及其mRNA表达均明显增高（P〈0．01），P450芳香化酶mRNA表达显著升高（P〈0．05，P〈0．01）。大鼠含药血清组：与对照组比较，补肾调经方中、高剂量组E2均升高（P〈0．01），cAMP明显升高（P〈0．05，P〈0．01），补肾调经方中剂量组P明显降低（P〈0．01）；补肾调经方中、高剂量组人卵巢颗粒细胞FSHR蛋白及其mRNA表达均增高（P〈0．01），P450芳香化酶mRNA表达明显升高（P〈0．05，P〈0．01）。结论补肾调经方可能通过调节卵巢颗粒细胞FSH／cAMP-PKA通路的主要效应分子FSHR、cAMP、P450arom、E2，促进卵巢颗粒细胞的内分泌功能。

Effect of Bushen Tiaojing Recipe Containing Serum on FSH/cAMP-PKA Pathway in in vitro Cul-tured Human Ovarian Granular Cells

Objective To explore the potential molecular mechanisms for Bushen Tiaojing Reci- pe （BTR） improving the endocrine function of ovarian granular cells by observing the effect of BTR con- taining serum on follicle stimulating hormone/cyclic adenosine monophosphate-protein kinase A （FSH/ cAMP-PKA） pathway in in vitro cultured human ovarian granular cells. Methods The primary ovarian granular cells collected from in vitro fertilization-embryo transfer patients were cultured for 24 h. The hu- man and rat serum containing different concentrations of BTR （low, medium, high dose）, and their nor- mal serums were co-incubated with ovarian granular cells for 48 h respectively, and then they were divid- ed into the low, medium, high dose BTR groups and the control group. The levels of estradiol （E2）, pro-gesterone （P）, and cyclic adenosine monophosphate （cAMP） in the culture medium were measured by radioimmunoassay. The protein expression of FSHR in ovarian granular cells was detected by Western Blot. The mRNA expression of follicle stimulating hormone receptor （FSHR） and P450 aromatase （P450arom） in ovarian granular cells were detected by Real-time PCR. Results In human BTR contai- ning serum groups： Compared with control group, the levels of E2 and cAMP in the culture medium were higher （both P 〈0.05 ）in the medium and high dose BTR groups; the levels of P in the culture medium de- creased in the medium dose BTR group （P 〈0.01）.The protein and mRNA expression of FSHR in ovarian granular cells increased （all P 〈 0.01 ）, the mRNA expressions of P450arom in ovarian granular cells were higher（P 〈0.05, P 〈0.01 ） in the medium and high dose BTR groups. In rat BTR containing serum groups： Compared with the control group, the levels of E2 in the culture medium were higher （all P 〈 0.01）, cAMP in the culture medium were higher （P 〈0.05, P 〈0.01 ） in the medium and high dose BTR group; the levels of P in the culture medium decreased in the medium dose BTR group（P 〈0.01 ）. The protein and mRNA expression of FSHR in ovarian granular cells were higher （all P 〈0.01 ）, the mRNA ex- pression of P450arom in ovarian granular cells increased in the medium and high dose BTR groups （P 〈 0.05, P 〈0.01 ）. Conclusion BTR could possibly improve the endocrine function of ovarian granular cells by regulating main effector molecules FSHR, cAMP, P450arom, and E2in FSH/cAMP-PKA pathway of ovarian granular cells.