抗苗勒管激素对卵巢黄素化颗粒细胞中芳香酶表达的影响

Effect of anti-Miillerian hormone on P450 aromatase mRNA expression in cultured human luteiulzed granulose cells

目的探讨抗苗勒管激素（AMH）对卵巢黄素化颗粒细胞激素分泌和芳香酶P450mRNA表达的影响。方法采集2006年6—12月在中山大学附属第二医院进行体外受精-胚胎移植（IVF-ET）的10例不孕患者的卵巢黄素化颗粒细胞进行原代培养，按照加入AMH浓度的不同，将颗粒细胞分为A、B、C、D、E组及睾酮对照组、空白对照组，A～E组分别加入1、5、10、20、50μg／L的AMH及1×10^4mol／L睾酮，睾酮对照组加入1×10^-7mol／L睾酮，空白对照组仅加入培养基。于培养24、48、72h时分别检测各组细胞中的雌二醇水平；培养72h后各组均进行细胞计数，并采用RT—PCR技术检测B、C、D、E组及睾酮对照组细胞中芳香酶P450mRNA的表达水平。结果（1）培养24、48、72h后颗粒细胞中的雌二醇水平，A组分别为（8．529±0．381）×10^4、（10．977±0．436）×10^4、（13．309±0．506）×10^4pmol／L，B组分别为（7．027±0．276）×10^4、（9．167±0．300）×10^4、（10．794±0．555）×10^4pmo]／L，C组分别为（6．039±0．226）×10^4、（7．585±0．548）×10^4、（8．797±0．518）×10^4pmol／L，D组分别为（5．118±0．460）×10^4、（5．716±0．496）×10^4、（6．205±0．667）×10^4pmo]／L，E组分别为（4．932±0．148）×10^4、（5．323±0．184）×10^4、（5．629±0．212）×10^4pmol／L，各组分别与空白对照组[分别为（0．001±0．001）×10^4、（0．006±0．003）×10^4、（0．029±0．011）×10^4pmol／L]比较，差异均有统计学意义（P〈0．01）；B、C、D、E组分别与睾酮对照组[分别为（8．418±0．569）×10^4、（10．841±0．689）×10^4、（13．301±0．637）×10^4pmol／L]比较，差异也均有统计学意义（P〈0．01）；B组分别与C、D、E组比较，C组分别与D、E组比较，差异也均有统计学意义（P〈0．01）；D组与E组之间比较，差异无统计学意义（P〉0．05）。A、B、C、D、E组及睾酮对照组培养24h后颗粒细胞中的雌二醇水平，分别与培养48、72h比较，差异均有统计学意义（P〈0．01）；培养48h后的雌二醇水平与培养72h比较，差异也均有统计学意义（P〈0．01）。空白对照组培养24、48、72h后的雌二醇水平比较，差异均无统计学意义（P〉0．05）。（2）培养72h后，B、C、D、E组颗粒细胞中芳香酶P450mRNA的表达水平分别为0．6148±0．0046、0．5156±0．0012、0．4698±0．0027、0．4282±0．0017，分别与睾酮对照组（0．8224±0．0021）比较，差异均有统计学意义（P〈0．01）；B组分别与C、D、E组比较，C组分别与D、E组比较，差异也有统计学意义（P〈0．01）；D组与E组之间比较，差异无统计学意义（P〉0．05）。结论AMH可能通过在卵巢局部抑制芳香酶活性而影响颗粒细胞的雌激素合成过程，促使卵泡内局部高雄激素环境的形成。

Objective To investigate the effect of anti-Mtillerian hormone （AMH） on hormone secretion and P450 aromatase mRNA expression from cultured human luteinized granulosa cells. Methods Human luteinized granulose cells were derived from 10 patients treated by in vitro fertilization-embryo transplantation （IVF-ET） in the Second Affiliated Hospital of Sun Yat-sen University from June to December 2006. Granulose cells were divided into group A, B, C, D, E depending on different concentration of AMH, testosterone group and blank group. 1 ～ 10^-7mol/L testosterone and 1,5,10,20,50 μg/L AMH were added into the culture medium of group A,B,C,D and E. 1 × 10^-7mol/L testosterone was added into the culture medium of testosterone group while no other ingredient was added into the medium of blank group. Estrogen levels in supemates were measured at 24,48,72 hours after cell incubation. RT-PCR was performed to detect the P450 aromatase mRNA expression in group B, C, D, E and testosterone group at 72 hours after cell incubation. Results （1） Estrogen levels in supernates of granulose cell culture at 24,48,72 hours were （8.529±0.381） ×10^4, （10.977 ±0.436） ×10^4, （13.309 ±0.506）×10^4 pmol/L in group A, （7.027 ± 0. 276）×10^4, （9. 167 ±0. 300） ×10^4, （ 10. 794 ±0. 555 ） ×10^4pmol/L in group B, （6. 039±0. 226）×10^4, （7.585±0.548）×10^4, （8.797 ±0.518） ×10^4 pmol/L in group C, （5. 118 ±0.460） ×10^4, （5.716 ± 0. 496） ×10^4, （6. 205 ±0. 667）×10^4pmol/L in group D, （4. 932 -±0. 148）×10^4, （5. 323 ±0. 184）×10^4, （5.629±0.212）×10^4 pmol/L in group E. When compared with blank group [（0.001±0.001） ×10^4, （0. 006 ±0. 003） ×10^4, （0. 029 ±0. 011 ） ×10^4 pmoL/L], the statistical differences were observed in group A,B,C, D,E（P 〈 0. 01 ） ; when compared with testosterone group [ （8. 418 ± 0. 569 ） ×10^4, （ 10. 841 ± 0. 689）×10^4, （ 13. 301 ±0. 637）×10^4pmol/L] , the statistical differences were observed in group B ,C ,D and E（ P 〈 0. 01 ） ; statistical differences were also observed in group C, D and E when compared with group B, and also group D and E when compared with group C（P 〈0.01 ）. No significant difference was observed between group D and E （P 〉 0. 05）. In group A, B, C, D,E and testosterone group, the estrogen levels at 24 hours after cell culture were significantly lower than those at 48 and 72 hours （ P 〈 0. 01 ） ; statistical difference was observed between estrogen levels at 48 and 72 hours（ P 〈 0. 01 ）. No significant difference was observed among 24,48 and 72 hours in blank group （ P 〉 0. 05 ）. （ 2 ） Relative ratios of intensity of P450 aromatase/β-actin at 72 hours of cell culture in group B, C, D and E were 0. 6148± 0. 0046, 0. 5156 ± 0. 0012, 0. 4698 ± 0. 0027 and 0. 4282 ± 0. 0017, respectively, which were statistically lower than that in testosterone group （0. 8224 ±0. 0021, P 〈0. 01 ）;statistical differences were also observed in group C, D and E when compared with group B, and also group D and E when compared with group C（P 〈0. 01 ）. No significant difference was observed between group D and E （ P 〉 0.05 ）. Conclusion It is suggested that AMH might affect estrogen synthesis by inhibiting P450 aromatose activity so that lead to hyperandrogenism microenvironment in local ovary.