性腺賀爾蒙、山藥及卵巢切除處理對子宮內膜細胞體外增殖 與子宮基質金屬蛋白質酶活性的影響

Abstract

背景與主題 適當子宮內膜對懷孕有重大影響。內膜受濾泡刺激素(FSH)，雌激素和黃體素影響。基因合成FSH(r-FSH)對內膜影響仍不清楚。砷(As)是自然致癌物可抑制細胞增殖及治療腫瘤。Benomyl (Ben)抑制有絲分裂應用於真菌防治。Ben可抑制細胞增殖，干擾有絲分裂及干擾內膜細胞。Carbendazim (Carb)具抗腫瘤活性，干擾有絲分裂及調節人體免疫。類固醇影響卵泡及內膜發展。類固醇可結合內膜受體及影響內膜發展。睾固酮具改變內膜形態和功能。睾固酮調節內膜性賀爾蒙激素接受體並抑制內膜增殖和分化。Louisianins A分離自鏈黴菌可顯著抑制癌細胞。Diosgenin是一種傳統山藥萃取物，使用於更年期婦女之賀爾蒙補充治療，上述試劑對內膜影響文獻並不多，本研究將偵測其影響並討論相關機轉。亦將探討補充diosgenin對於切除卵巢母鼠子宮內膜MMP-2及-9 之表現影響‧材料與方法收集子宮內膜刮除術病患之檢體。將內膜細胞分離培養，根據r-FSH (Gona-F,Puregon)濃度將細胞分組：(1) 0; (2) 1; (3) 10; (4) 100; (5) 1000; (6) 100000 μIU/ml‧根據黃 體素濃度分組：(1) 0; (2) 1; (3) 10; (4) 100 μg/ml‧根據As,Ben,Carb濃度分組：(1) 0 M;(2) 10-6 M; (3) 10-5 M; (4) 10-4 M。根據類固醇，睾固酮濃度分組：(1) 0, (2) 0.01; (3) 0.1;(4) 1 μM。根據Louisianins A濃度分組：(1) 0 mM, (2) 0.01; (3) 0.05; (4) 0.1; (5) 1; (6) 5;(7) 10 mM。經48小時培養，使用MTT及ELISA檢測細胞吸收度以偵測其對內膜影響‧7週大切除卵巢之母鼠或控制組老鼠，使用不同劑量之diosgenin (0, 10, 50, 100 mg/kg/day)達8週後，抽取其血清progesterone (P4)及estradiol (E2)數值，並切取子宮以進一步分析子宮肌層內MMP-2，-9及collagen之表現‧研究結果Gonal-F組經24/48小時吸收度為：(1) 100/100; (2) 103.8/102.3; (3) 104.8/102.8; (4)102.3/101.3; (5) 96.3/94.2; (6) 86.8/84.3%。Puregon組吸收度是：(1) 100/100; (2)102.8/101.9; (3) 103/102.3; (4) 103.9/103.5; (5) 102.9/102.4; (6) 103.7/103%。低或中劑量 Gonal-F對內膜無顯著影響。高劑量Gonal-F(100000 μIU/ml)抑制內膜‧Puregon不顯著影響內膜，黃體素組吸收度是：(1) 100/100; (2) 99.1/101.9; (3) 83.5/80.4; (4) 80.7/82.4%‧高劑量黃體素(10, 100 μg/ml)明顯抑制內膜‧頭24小時As，Ben，Carb對內膜無顯著影響。48小時後高劑量As，Ben，Carb明顯抑制內膜。As組經24/48小時培養後吸收度：(1) 100/100; (2) 104.8/82.1; (3) 110.7/43.6; (4) 108.8/35.3%。Ben組吸收度：(1) 100/100; (2) 97.5/75.9; (3) 95.4/66.4; (4) 101.2/49.6%‧As，Ben濃度與內膜增殖呈負相關。高劑量As及Ben明顯抑制內膜。Carb組吸收度：(1)100/100; (2) 131.3/70.4; (3) 103.1/73; (4) 82.7/76.7%‧Carb濃度為1，10 ，100x10-6 M明顯抑制內膜‧Dexamethasone組吸收度：(1) 100; (2) 106.1; (3) 95.5; (4) 87.8%‧低及中劑量Dexamethasone不影響內膜‧高劑量(1uM) 顯著抑制內膜。睾固酮組吸收度為：(1) 100";(2) 107.6; (3) 112.5; (4) 110.1%。低劑量睾固酮不顯著影響內膜。高劑量(0.1, 1 μM)刺激內膜。Louisianins組吸收度為：(1) 100; (2) 98.5; (3) 100.4; (4) 102.7; (5) 64.8; (6) 48.6; (7)19.4 %。低劑量Louisianins不影響內膜。高劑量(1, 5, 10 mM)抑制內膜。 結果顯示切除卵巢8週後的老鼠體重較重，經diosgenin補充後，切除卵巢與否對於老鼠體重無明顯影響‧切除卵巢母鼠之P4及E2濃度比控制組為低‧補充diosgenin可提高控制組母鼠P4濃度，切除卵巢母鼠MMP-2及-9 mRNA表現較控制組為低，經由diosgenin補充後可提高MMP-2及下降-9 mRNA表現‧切除卵巢母鼠之collagen mRNA表現比控制組為高‧經diosgenin補充後可降低collagen mRNA表現‧ 結 論 總結而言，不同r-FSH對內膜有不同影響。低劑量Gonal-F，Puregon和黃體素對內膜無顯著影響。48小時培養，高劑量Gonal -F (100000 μIU/ml)和黃體素(10, 100 μg/ml)抑制內膜增殖‧Puregon無此抑制現象。高濃度As，Ben，Carb，Dexamethasone和Louisianins 明顯抑制內膜‧高劑量睾固酮刺激內膜生長，r -FSH對內膜影響差異是α和β分子結構和效價及內膜接受體差異所造成。這些物質影響內膜及子宮接受能力。不孕症病患需注意污染食物和水造成不良影響‧Diosgenin與切除卵巢母鼠之gelatinase及collagen表現有關，diosgenin補充可改善切除卵巢對於gelatinase表現及賀爾蒙濃度之負面影響‧適量diosgenin 補充可改善子宮肌層內gelatinase及collagen之表現‧本研究對賀爾蒙、類固醇,化學因子,gelatinase及collagen對子宮內膜影響提供初步數據及研究方向。

Background and subject Adequate endometrial development plays significant roles in pregnancy. Endometrial changes are controlled by recombinant-FSH (r-FSH), estrogen and progesterone. Arsenic (As), a natural carcinogen, are inhibitors of cell proliferation and used in tumor therapy. Benomyl (Ben) could inhibit cell proliferation and blocked mitosis and endometrial growth. Carbendazim (Carb) could inhibit tumor, interfere mitosis, and down-regulate human immunity. Steroid influence the folloculogenesis and endometrial cells. Dexamethosone could bind to endometrial receptors and influence endometrial development. Androgen plays important roles in morphological and functional changes of endometrium. Testosterone regulates sex hormone receptor expression in endometrium and influence endometrial proliferation and differentiation. Louisianins A, isolated from Streptomyces s, remarkably inhibited the tumor growth. Diosgenin, a traditional Yam extraction, has been used in hormone replacement for menopausal women. Few investigators demonstrated their influences upon endometrium. Herein we planed to investigate their roles upon endometrial proliferation. We also tried to investigate the influences of diosgenin administration upon the MMP-2 and -9 activity and expression and reproductive hormones of ovariectomized (OVX) rats, a model of menopausal status.

Materials and Methods Women accepting dilation & curettage of uterus were recruited. Endometrial cells were isolated and cultured. According as r-FSH (Gona-F, Puregon) concentrations, endometrial cells were divided: (1) 0; (2) 1; (3) 10; (4) 100; (5) 1000; (6) 100000 μIU/ml. According as progesterone concentrations, cells were divided: (1) 0; (2) 1; (3) 10; (4) 100 μg/ml. According as As, Ben, and Carb concentrations, cells were divided: (1) 0 M; (2) 10-6 M; (3) 10-5 M; (4) 10-4 M. According as dexamethasone and testosterone concentrations, cells were divided: (1) 0, (2) 0.01; (3) 0.1; (4) 1 μM. According as Louisianins A concentrations, cells were divided: (1) 0, (2) 0.01; (3) 0.05; (4) 0.1; (5) 1; (6) 5; (7) 10 mM. After 48-hour culture, endometrial cell proliferations were assessed by dimethylthiazol-diphenyltetrazolium bromide (MTT) and enzyme-linked immunosorbent assay (ELISA) plate reader. The influences of different agents and dosages upon endometrial cell proliferation were compared. Seven-week old female Wistar rats with bilateral OVX or sham operation (controls) were divided and administered different dosages of diosgenin (0, 10, 50, or 100 mg/kg/day) for 8 weeks. Serum was sampled for progesterone (P4) and estradiol (E2) assay and uterine horns harvested. Myometrial MMP-2 and -9 activity and expression were surveyed and myometrial collagen expression was also assayed.

Results The cell absorption of Gonal-F groups after 24/48 hours culture were: (1) 100/100; (2) 103.8/102.3; (3) 104.8/102.8; (4) 102.3/101.3; (5) 96.3/94.2; (6) 86.8/84.3%.The absorption of Puregon group were: (1) 100/100; (2) 102.8/101.9; (3) 103/102.3; (4) 103.9/103.5; (5) 102.9/102.4; (6) 103.7/103.2%. Low or moderate-dosage Gonal-F appeared non-significant influences upon endometrial growth. High dosage Gonal-F (100000 μIU/ml) inhibit endometrial cells. Puregon appeared non-significant influence upon endometrium. The cell absorption of progesterone groups were: (1) 100/100; (2) 99.1/101.9; (3) 83.5/80.4; (4) 80.7/82.4%. Higher-dosage progesterone (10, 100 μg/ml) significantly inhibits endometrial cells. During first 24 hours, As, Ben and Carb non-significantly influence endometrial growth. After 48 hour culture, higher-dosage As, Ben and Carb significantly inhibit endometrial growth. In As groups, the cell absorption after 24/48-hour culture were: (1) 100/100; (2) 104.8/82.1; (3) 110.7/43.6; (4) 108.8/35.3%. In Ben groups, the absorption were: (1) 100/100; (2) 97.5/75.9; (3) 95.4/66.4; (4) 101.2/49.6%. As and Ben appeared the dose-dependent inhibition upon endometrium. High-dose As and Ben significantly inhibit endometrium proliferations. The cell absorption of Carb groups were: (1) 100/100; (2) 131.3/70.4; (3) 103.1/73; (4) 82.7/76.7%. The existence of Carb (1, 10, 100x10-6 M) significantly inhibits endometrial proliferation. The cell absorptions of dexamethasone groups were: (1) 100; (2) 106.1; (3) 95.5; (4) 87.8%. Low and mid-dosage dexamethasone non-significantly influence the endometrial growth. High dexamethasone dosage (1 μM) significantly inhibit endometrial growth. In testosterone groups, the cell absorption for 4 groups were: (1) 100 ; (2) 107.6; (3) 112.5; (4) 110.1%. Low-dosage testosterone non-significantly influence the endometrial growth. High-dosage testosterone (0.1, 1 μM) stimulate endometrial growth. The cell absorption for Louisianins A were: (1) 100; (2) 98.5; (3) 100.4; (4) 102.7; (5) 64.8; (6) 48.6; (7) 19.4% . Low–dosages Louisianins A doesn’t influence endometrial growth. High -dosages Louisianins A (1, 5, 10 mM) appeared the inhibition effects. The results show higher body weight in OVX rats across the 8 weeks post surgery. No significant differences were noted among OVX or Sham rats with diosgenin supplements. There were lower P4 and E2 concentrations in OVX rats compared to Sham rats, and higher P4 concentration of Sham rats post diosgenin supplement. MMP-2 and -9 mRNA expression and activity was lower in OVX rats, although higher MMP-2 and lower MMP-9 activity/mRNA expression was observed in OVX rats post diosgenin supplementation. Collagen mRNA expression was higher in OVX rats compared to Sham controls, and diosgenin administration decreased collagen mRNA expression in OVX rats. Conclusions In conclusion, different r-FSH appears different influence upon endometrial growth. Lower-dosage Gonal-F, Puregon, and progesterone non-significantly influence endometrial growth. High-dosage Gonal-F (100000 μIU/ml) and progesterone (10, 100 μg/ml), but not Puregon, inhibit endometrial growth. High-dosage As, Ben, Carb, dexamethasone and Louisianins A significantly inhibit endometrial proliferation. High-concentration testosterone stimulate endometrial growth. The differences of r-FSH upon endometrium might be due to the differentα and β formulations, molecular structure, potencies and receptors. They might influence endometrial growth and uterine receptivity. It also suggested the necessity of prevention of contaminated food and water prior and during ART protocols, especially for the individuals with thinner endometrium. Diosgenin is associated with gelatinase expression and collagen metabolism in OVX rats. Diosgenin administration can partially reverse the effects of OVX upon MMP functions and hormone status. Adequate diosgenin supplement might modulate myometrial gelatinase expression and collagen metabolism in menopausal subjects. This study provided the preliminary database for the related surveys and future direction.