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1×10-7mol/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、72 h时分别检测各组细胞中的雌二醇水平;培养72 h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P450 mRNA的表达水平.结果 (1)培养24、48、72 h后颗粒细胞中的雌二醇水平,A组分别为(8.529±0.381)×104、(10.977±0.436)x 104、(13.309±0.506)×104pmo/L,B组分别为(7.027±0.276)×104、(9.167±0.300)×104、(10.794±0.555)×104 pmo/L,C组分别为(6.039±0.226)×104、(7.585±0.548)×104、(8.797±0.518)×104pmo/L,D组分别为(5.118±0.460)×104、(5.716±0.496)×104(6.205±0.667)x1044pmol/L,E组分别为(4.932±0.148)×104(5.323±0.184)×104(5.629±0.212)x 1044 pmol/L,各组分别与空白对照组[分别为(0.001±0.001)×104(0.006±0.003)×104(0.029±0.011)×1044pmol/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)×104(10.841±0.689)×104(13.301±0.637)×1044pmol/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(P40.05).A、B、C、D、E组及睾酮对照组培养24 h后颗粒细胞中的雌二醇水平,分别与培养48、72 h比较,差异均有统计学意义(P<0.01);培养48 h后的雌二醇水平与培养72 h比较,差异也均有统计学意义(P<0.01).空白对照组培养24、48、72 h后的雌二醇水平比较,差异均无统计学意义(P>0.05).(2)培养72 h后,B、C、 D、 E组颗粒细胞中芳香酶P450 mRNA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.
目的 探討抗苗勒管激素(AMH)對卵巢黃素化顆粒細胞激素分泌和芳香酶P450mRNA錶達的影響.方法 採集2006年6-12月在中山大學附屬第二醫院進行體外受精-胚胎移植(IVF-ET)的10例不孕患者的卵巢黃素化顆粒細胞進行原代培養,按照加入AMH濃度的不同,將顆粒細胞分為A、B、C、D、E組及睪酮對照組、空白對照組,A～E組分彆加入1、5、10、20、50μg/L的AMH及1×10-7mol/L睪酮,睪酮對照組加入1×10-7mol/L睪酮,空白對照組僅加入培養基.于培養24、48、72 h時分彆檢測各組細胞中的雌二醇水平;培養72 h後各組均進行細胞計數,併採用RT-PCR技術檢測B、C、D、 E組及睪酮對照組細胞中芳香酶P450 mRNA的錶達水平.結果 (1)培養24、48、72 h後顆粒細胞中的雌二醇水平,A組分彆為(8.529±0.381)×104、(10.977±0.436)x 104、(13.309±0.506)×104pmo/L,B組分彆為(7.027±0.276)×104、(9.167±0.300)×104、(10.794±0.555)×104 pmo/L,C組分彆為(6.039±0.226)×104、(7.585±0.548)×104、(8.797±0.518)×104pmo/L,D組分彆為(5.118±0.460)×104、(5.716±0.496)×104(6.205±0.667)x1044pmol/L,E組分彆為(4.932±0.148)×104(5.323±0.184)×104(5.629±0.212)x 1044 pmol/L,各組分彆與空白對照組[分彆為(0.001±0.001)×104(0.006±0.003)×104(0.029±0.011)×1044pmol/L]比較,差異均有統計學意義(P<0.01);B、C、D、E組分彆與睪酮對照組[分彆為(8.418±0.569)×104(10.841±0.689)×104(13.301±0.637)×1044pmol/L]比較,差異也均有統計學意義(P<0.01);B組分彆與C、D、E組比較,C組分彆與D、E組比較,差異也均有統計學意義(P<0.01);D組與E組之間比較,差異無統計學意義(P40.05).A、B、C、D、E組及睪酮對照組培養24 h後顆粒細胞中的雌二醇水平,分彆與培養48、72 h比較,差異均有統計學意義(P<0.01);培養48 h後的雌二醇水平與培養72 h比較,差異也均有統計學意義(P<0.01).空白對照組培養24、48、72 h後的雌二醇水平比較,差異均無統計學意義(P>0.05).(2)培養72 h後,B、C、 D、 E組顆粒細胞中芳香酶P450 mRNA的錶達水平分彆為0.6148±0.0046、0.5156±0.0012、0.4698±0.0027、0.4282±0.0017,分彆與睪酮對照組(0.8224±0.0021)比較,差異均有統計學意義(P<0.01);B組分彆與C、D、E組比較,C組分彆與D、E組比較,差異也有統計學意義(P<0.01);D組與E組之間比較,差異無統計學意義(P>0.05).結論AMH可能通過在卵巢跼部抑製芳香酶活性而影響顆粒細胞的雌激素閤成過程,促使卵泡內跼部高雄激素環境的形成.
목적 탐토항묘륵관격소(AMH)대란소황소화과립세포격소분비화방향매P450mRNA표체적영향.방법 채집2006년6-12월재중산대학부속제이의원진행체외수정-배태이식(IVF-ET)적10례불잉환자적란소황소화과립세포진행원대배양,안조가입AMH농도적불동,장과립세포분위A、B、C、D、E조급고동대조조、공백대조조,A～E조분별가입1、5、10、20、50μg/L적AMH급1×10-7mol/L고동,고동대조조가입1×10-7mol/L고동,공백대조조부가입배양기.우배양24、48、72 h시분별검측각조세포중적자이순수평;배양72 h후각조균진행세포계수,병채용RT-PCR기술검측B、C、D、 E조급고동대조조세포중방향매P450 mRNA적표체수평.결과 (1)배양24、48、72 h후과립세포중적자이순수평,A조분별위(8.529±0.381)×104、(10.977±0.436)x 104、(13.309±0.506)×104pmo/L,B조분별위(7.027±0.276)×104、(9.167±0.300)×104、(10.794±0.555)×104 pmo/L,C조분별위(6.039±0.226)×104、(7.585±0.548)×104、(8.797±0.518)×104pmo/L,D조분별위(5.118±0.460)×104、(5.716±0.496)×104(6.205±0.667)x1044pmol/L,E조분별위(4.932±0.148)×104(5.323±0.184)×104(5.629±0.212)x 1044 pmol/L,각조분별여공백대조조[분별위(0.001±0.001)×104(0.006±0.003)×104(0.029±0.011)×1044pmol/L]비교,차이균유통계학의의(P<0.01);B、C、D、E조분별여고동대조조[분별위(8.418±0.569)×104(10.841±0.689)×104(13.301±0.637)×1044pmol/L]비교,차이야균유통계학의의(P<0.01);B조분별여C、D、E조비교,C조분별여D、E조비교,차이야균유통계학의의(P<0.01);D조여E조지간비교,차이무통계학의의(P40.05).A、B、C、D、E조급고동대조조배양24 h후과립세포중적자이순수평,분별여배양48、72 h비교,차이균유통계학의의(P<0.01);배양48 h후적자이순수평여배양72 h비교,차이야균유통계학의의(P<0.01).공백대조조배양24、48、72 h후적자이순수평비교,차이균무통계학의의(P>0.05).(2)배양72 h후,B、C、 D、 E조과립세포중방향매P450 mRNA적표체수평분별위0.6148±0.0046、0.5156±0.0012、0.4698±0.0027、0.4282±0.0017,분별여고동대조조(0.8224±0.0021)비교,차이균유통계학의의(P<0.01);B조분별여C、D、E조비교,C조분별여D、E조비교,차이야유통계학의의(P<0.01);D조여E조지간비교,차이무통계학의의(P>0.05).결론AMH가능통과재란소국부억제방향매활성이영향과립세포적자격소합성과정,촉사란포내국부고웅격소배경적형성.
Objective To investigate the effect of anti-Mlllerian 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 supernates 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)×104, (10.977±0.436)×104, (13.309±0.506)×104 pmol/L in group A, (7.027±0.276)×104, (9.167±0.300)×104, (10.794±0.555)×104 pmol/L in group B, (6.039±0.226)×104,(7.585±0.548)×104, (8.797±0.518)×104 pmol/L in group C, (5.118±0.460)×104, (5.716±0.496)×104, (6.205±0.667)×104 pmol/L in group D, (4.932±0.148)×104, (5.323±0.184)×104,(5.629±0.212)×104 pmol/L in group E. When compared with blank group [(0.001±0.001)×104,(0.006±0.003)×104, (0.029±0.011)×104 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)×104, (10.841±0.689)×104, (13.301±0.637)×104 pmol/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 at72 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.
