PCOS模型大鼠P450芳香化酶表达的研究
摘要
目的:比较DHEA+Ins与HCG+Ins两种方法构建的PCOS大鼠模型。研究PCOS大鼠卵巢、子宫、肌肉、脂肪组织中P450 arom的表达水平。探讨不同的芳香化酶抑制剂(来曲唑)给药方案治疗PCOS大鼠的疗效。
方法: 42d龄SD大鼠80只,随机分为四组:模型组Ⅰ45只大鼠使用DHEA+Ins造模,模型组Ⅱ15只大鼠使用HCG+Ins造模,分别设置相应对照组各10只大鼠。各组大鼠均于70d龄时开始连续阴道涂片20天观察动情周期变化,90d龄检测血清总T、E_2、P、LH、FSH、FPG、FINS水平,计算LH/FSH及Homa-IR。从两模型组中各取10只无动情周期的大鼠,摘取双侧卵巢,一侧卵巢行组织学切片,另一侧卵巢用于测定P450 arom(RT-PCR技术)的表达水平。取肌肉、脂肪组织、子宫用于测定其P450 arom(RT-PCR技术)的表达水平。从模型组I中选择无动情周期大鼠30只随机分为三组,对照组10只,治疗组Ⅰ10只大鼠给来曲唑1.4mg/100mg·d灌胃,治疗组Ⅱ10只大鼠给来曲唑11mg/100mg·4d灌胃,20天后取血,检测血清总T、E_2、P、LH、FSH、FPG、FINS水平,计算LH/FSH及Homa-IR。
结果:(1)模型组Ⅰ、Ⅱ中失去规律动情周期的大鼠的比例分别为95.6%、93.3%,两者无显著差异(P>0.05);两组大鼠卵巢重量均显著高于相应对照组;两组大鼠卵巢均可见多个扩张的囊泡,平均囊泡直径组Ⅰ(0.048±0.012)显著小于组Ⅱ(0.069±0.036),黄体计数组Ⅰ(0.10±0.32)显著少于组Ⅱ(0.70±0.82);血清P水平模型组Ⅰ(14.38±2.92)与模型组Ⅱ(17.28±3.12)无显著差异(P>0.05);两模型组与相应对照组大鼠相比较血清T水平、LH/FSH比值均有显著性升高(P<0.05),P均显著降低(P<0.05),E2升高不明显(P>0.05); HOMA-IR模型组Ⅰ(8.89±0.86)显著高于模型组Ⅱ(6.86±0.48) (P<0.05)。(2)实验组大鼠肌肉、脂肪组织中P450 arom mRNA表达的相对含量与相应对照组均无显著差异;卵巢中实验组(2.80±1.19)与对照组(0.82±0.18)差异有显著性(P<0.05);子宫中实验组(1.83±0.40)与对照组(0.58±0.10)差异有显著性(P<0.05)。(3)组内比较两治疗组给药后E_2、P均显著升高(P<0.05), LH/FSH、FINS、Homa-IR均显著降低(P<0.05),但T、FPG无显著差异(P>0.05);组间比较治疗组Ⅰ与治疗组Ⅱ给药20天后血清T、E_2、P、LH、FSH、均无显著差异(P>0.05),Homa-IR治疗组Ⅰ(4.51±0.35)显著低于治疗组Ⅱ(5.11±0.43)。
结论: (1) DHEA+Ins构建的PCOS大鼠模型同时具有排卵障碍、高雄激素血症、卵巢囊性变及胰岛素抵抗等PCOS的典型特征,且具有一定的稳定性。(2) PCOS大鼠卵巢及子宫中P450 arom表达增高,且卵巢中P450 arom的表达水平与胰岛素抵抗的发生呈正相关。(3)来曲唑能有效治疗PCOS大鼠的排卵障碍。而且没有造成PCOS大鼠血清T水平升高。连续给药或单次给药的促排卵效果无显著差异,但连续给药的方法更适用于治疗伴有胰岛素抵抗的PCOS患者。
Objective: Making a model of PCOS rats, we can compare the P450 aromatase expression of PCOS rats and the normal ones, in their ovaries and uteruses, also in muscle and fat. Furthermore, we can probe into the effection of which two different administrator of arimedex (letrozole) while treating PCOS.
Method: randomizing 80 SD rats of 42 days into 4 groups: group I---45 rats modeled in DHEA + Ins, group II---15 rats modeled in HCG + Ins, two corresponding control groups of 10 rats respectively. When the rats are at 70 days, we give them 20 days of continuous vaginal smear, observing the variation of their estrous cycle. While at 90 days, the degree of their T, E_2, P, LH, FSH, FPG, FINS in the serum should be determined, also the LH/FSH and Homa-IR. Selected 10 rats which have not regular estrous cycle, we excise their bilateral ovaries, one side with histological slice, and the other side used for the determination of mRNA in P450 aromatase. We also excise the muscle, fatty tissue and uterus, which are also used for the determination that we have mentioned above. Choosing the rats (PCOS rats) which are modeled successfully, we randomize them into two treating groups, group I---10 rats treated with intragastric administration of letrozole in the dosage of 1.4mg/100mg·d, group II---10 rats, the same treatment as group I, but in the dosage of 11mg/100mg·4d.
Results: (1) There were 95.6 percent of rats losting regular estrous cycle in the model I and 93.3 percent of rats in the model II. The ovary weight of rats in both two models was heavier than that of controls. Rats of both two models showed polycystic ovary-like syndrome manifestation, The average saccular ovulas in model I (0.048±0.012) was smaller than those in model II (0.069±0.036), The number of corpora lutea in model I (0.10±0.32) was smaller than that in model II (0.70±0.82). Serum levels of T, LH/FSH increased in both two models,P decreased in both two models, Serum levels of E_2 had no significant increase (P>0.05). HOMA-IR of both two models were higher than that of controls with model I (8.89±0.86) significantly higher than model II (6.86±0.48). (2) While determining the amount of mRNA expression of P450 aromatase in the rats’muscle, we find out that the modeling group (0.42±0.04) is nearly as the same as its control group (0.38±0.04). In the rats’fat tissue, the modeling group (0.45±0.05) is also as the same as its control group (0.38±0.05). In the rats’ovary, the modeling group (2.80±1.19) is significantly higher than its control group (0.82±0.18). In the rats’uterus, the modeling group (1.83±0.40) is significantly higher than its control group (0.58±0.10). (3) After administration of the two treating groups, we can find out that the degrees of E_2 and P are significant higher than that before administration (P<0.05). The LH/FSH and FINS are significant lower than that before administration. T and FPG have no remarkable difference while compared with that before administration (P>0.05). We determined the T, E_2, P, LH, FSH in the serum, that of the treated group I have no significant difference with that of the treated group II (P<0.05), but the degree of HOMA-IR of the treated group I (4.51±0.35) are remarkable lower than that of the treated group II (5.11±0.43).
Conclusion: (1) The model of DHEA+Ins is more similar to human PCOS in the morphology of ovary and the degree of insulin resistance,and it is more stable. (2) The amount of mRNA expression of P450 aromatase in rats’uteruses and ovaries would increase, if the PCOS rats are modeled in DHEA + Ins. The expression of P450 arom in PCOS rats is positive correlation with HOMA-IR. (3) Arimedex (letrozole) can induce the PCOS rats to ovulate effectively without the increasing of T. The different methods of administration have no influence on the serum endocrine, but the method of 1.4mg/100mg·d can lower the insulin resistance effectively.
方法: 42d龄SD大鼠80只,随机分为四组:模型组Ⅰ45只大鼠使用DHEA+Ins造模,模型组Ⅱ15只大鼠使用HCG+Ins造模,分别设置相应对照组各10只大鼠。各组大鼠均于70d龄时开始连续阴道涂片20天观察动情周期变化,90d龄检测血清总T、E_2、P、LH、FSH、FPG、FINS水平,计算LH/FSH及Homa-IR。从两模型组中各取10只无动情周期的大鼠,摘取双侧卵巢,一侧卵巢行组织学切片,另一侧卵巢用于测定P450 arom(RT-PCR技术)的表达水平。取肌肉、脂肪组织、子宫用于测定其P450 arom(RT-PCR技术)的表达水平。从模型组I中选择无动情周期大鼠30只随机分为三组,对照组10只,治疗组Ⅰ10只大鼠给来曲唑1.4mg/100mg·d灌胃,治疗组Ⅱ10只大鼠给来曲唑11mg/100mg·4d灌胃,20天后取血,检测血清总T、E_2、P、LH、FSH、FPG、FINS水平,计算LH/FSH及Homa-IR。
结果:(1)模型组Ⅰ、Ⅱ中失去规律动情周期的大鼠的比例分别为95.6%、93.3%,两者无显著差异(P>0.05);两组大鼠卵巢重量均显著高于相应对照组;两组大鼠卵巢均可见多个扩张的囊泡,平均囊泡直径组Ⅰ(0.048±0.012)显著小于组Ⅱ(0.069±0.036),黄体计数组Ⅰ(0.10±0.32)显著少于组Ⅱ(0.70±0.82);血清P水平模型组Ⅰ(14.38±2.92)与模型组Ⅱ(17.28±3.12)无显著差异(P>0.05);两模型组与相应对照组大鼠相比较血清T水平、LH/FSH比值均有显著性升高(P<0.05),P均显著降低(P<0.05),E2升高不明显(P>0.05); HOMA-IR模型组Ⅰ(8.89±0.86)显著高于模型组Ⅱ(6.86±0.48) (P<0.05)。(2)实验组大鼠肌肉、脂肪组织中P450 arom mRNA表达的相对含量与相应对照组均无显著差异;卵巢中实验组(2.80±1.19)与对照组(0.82±0.18)差异有显著性(P<0.05);子宫中实验组(1.83±0.40)与对照组(0.58±0.10)差异有显著性(P<0.05)。(3)组内比较两治疗组给药后E_2、P均显著升高(P<0.05), LH/FSH、FINS、Homa-IR均显著降低(P<0.05),但T、FPG无显著差异(P>0.05);组间比较治疗组Ⅰ与治疗组Ⅱ给药20天后血清T、E_2、P、LH、FSH、均无显著差异(P>0.05),Homa-IR治疗组Ⅰ(4.51±0.35)显著低于治疗组Ⅱ(5.11±0.43)。
结论: (1) DHEA+Ins构建的PCOS大鼠模型同时具有排卵障碍、高雄激素血症、卵巢囊性变及胰岛素抵抗等PCOS的典型特征,且具有一定的稳定性。(2) PCOS大鼠卵巢及子宫中P450 arom表达增高,且卵巢中P450 arom的表达水平与胰岛素抵抗的发生呈正相关。(3)来曲唑能有效治疗PCOS大鼠的排卵障碍。而且没有造成PCOS大鼠血清T水平升高。连续给药或单次给药的促排卵效果无显著差异,但连续给药的方法更适用于治疗伴有胰岛素抵抗的PCOS患者。
Objective: Making a model of PCOS rats, we can compare the P450 aromatase expression of PCOS rats and the normal ones, in their ovaries and uteruses, also in muscle and fat. Furthermore, we can probe into the effection of which two different administrator of arimedex (letrozole) while treating PCOS.
Method: randomizing 80 SD rats of 42 days into 4 groups: group I---45 rats modeled in DHEA + Ins, group II---15 rats modeled in HCG + Ins, two corresponding control groups of 10 rats respectively. When the rats are at 70 days, we give them 20 days of continuous vaginal smear, observing the variation of their estrous cycle. While at 90 days, the degree of their T, E_2, P, LH, FSH, FPG, FINS in the serum should be determined, also the LH/FSH and Homa-IR. Selected 10 rats which have not regular estrous cycle, we excise their bilateral ovaries, one side with histological slice, and the other side used for the determination of mRNA in P450 aromatase. We also excise the muscle, fatty tissue and uterus, which are also used for the determination that we have mentioned above. Choosing the rats (PCOS rats) which are modeled successfully, we randomize them into two treating groups, group I---10 rats treated with intragastric administration of letrozole in the dosage of 1.4mg/100mg·d, group II---10 rats, the same treatment as group I, but in the dosage of 11mg/100mg·4d.
Results: (1) There were 95.6 percent of rats losting regular estrous cycle in the model I and 93.3 percent of rats in the model II. The ovary weight of rats in both two models was heavier than that of controls. Rats of both two models showed polycystic ovary-like syndrome manifestation, The average saccular ovulas in model I (0.048±0.012) was smaller than those in model II (0.069±0.036), The number of corpora lutea in model I (0.10±0.32) was smaller than that in model II (0.70±0.82). Serum levels of T, LH/FSH increased in both two models,P decreased in both two models, Serum levels of E_2 had no significant increase (P>0.05). HOMA-IR of both two models were higher than that of controls with model I (8.89±0.86) significantly higher than model II (6.86±0.48). (2) While determining the amount of mRNA expression of P450 aromatase in the rats’muscle, we find out that the modeling group (0.42±0.04) is nearly as the same as its control group (0.38±0.04). In the rats’fat tissue, the modeling group (0.45±0.05) is also as the same as its control group (0.38±0.05). In the rats’ovary, the modeling group (2.80±1.19) is significantly higher than its control group (0.82±0.18). In the rats’uterus, the modeling group (1.83±0.40) is significantly higher than its control group (0.58±0.10). (3) After administration of the two treating groups, we can find out that the degrees of E_2 and P are significant higher than that before administration (P<0.05). The LH/FSH and FINS are significant lower than that before administration. T and FPG have no remarkable difference while compared with that before administration (P>0.05). We determined the T, E_2, P, LH, FSH in the serum, that of the treated group I have no significant difference with that of the treated group II (P<0.05), but the degree of HOMA-IR of the treated group I (4.51±0.35) are remarkable lower than that of the treated group II (5.11±0.43).
Conclusion: (1) The model of DHEA+Ins is more similar to human PCOS in the morphology of ovary and the degree of insulin resistance,and it is more stable. (2) The amount of mRNA expression of P450 aromatase in rats’uteruses and ovaries would increase, if the PCOS rats are modeled in DHEA + Ins. The expression of P450 arom in PCOS rats is positive correlation with HOMA-IR. (3) Arimedex (letrozole) can induce the PCOS rats to ovulate effectively without the increasing of T. The different methods of administration have no influence on the serum endocrine, but the method of 1.4mg/100mg·d can lower the insulin resistance effectively.
引文
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[3] Vincent H, Helene B, Herve M, et al. Differential Regulation of Two 3' End Variants of P450 Aromatase Transcripts and of a New Truncated Aromatase Protein in Rabbit Preovulatory Granulosa Cells[J]. Endocrinology. 2003, 144(11): 4790-4798
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[8] Gersevich S, Poutanen M, Tapanainen J, et al. Hormonal regulation of rat 17 beta-hydroxysteroid dehydrogenate type 1 in cultured rat granulose cells: effects of recombinant follicle- stimulating hormone, estrogens, androgens, and epidermal growth factor[J]. Endocrinology.1994,135(5):1963
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[1] Iwabuchi J, Wako S, Tanaka T, et al. Analysis of the p450 aromatase gene expression in the Xenopus brain and gonad[J]. Steroid Biochem Mol Biol. 2007, 107(3-5):149-155
[2] Carreau S,Bourguiba S,Lambard S,et a1.Reproductive system:aromatase and estrogens[J].Mol Cell Endocrinol. 2002,193(1~2):137-143
[3] Vincent H, Helene B, Herve M, et al. Differential Regulation of Two 3' End Variants of P450 Aromatase Transcripts and of a New Truncated Aromatase Protein in Rabbit Preovulatory Granulosa Cells[J]. Endocrinology. 2003, 144(11): 4790-4798
[4] Ailawadi RK, Jobanputra S, Kataria M, et al. Treatment of endometriosis and chronic pelvic pain with letrozole and norethindrone acetate: a pilot study[J]. Fertil Steril.2004, 81:290-296
[5] Harlow CR, Bradshaw AC, Rae MT, et al. Oestrogen formation and connective tissue growth factor expression in rat granulosa cells[J]. Endocrinol. 2007, 192(1):41-52
[6] Klinghoffer RA, Mueting-Nelsen PF, Faerman A, et al. The two PDGF receptors maintain conserved signaling in vivo despite divergent embryological functions [J]. Mol Cell.2001,7(2):343-354
[7] Sirianni R, Chimento A, Malirindi R, et al. Insulin-like growth factor-1, regulating aromatase expression through steroidogenic factor 1, supports estrogen-dependent tumor Leydig cell proliferation[J]. Cancer Res. 2007, 67(17):8368-8377
[8] Gersevich S, Poutanen M, Tapanainen J, et al. Hormonal regulation of rat 17 beta-hydroxysteroid dehydrogenate type 1 in cultured rat granulose cells: effects of recombinant follicle- stimulating hormone, estrogens, androgens, and epidermal growth factor[J]. Endocrinology.1994,135(5):1963
[9] Lorenzo PL, Liu IK, et al. Influence of epidermal growth factor on manmalianoocyte maturation via tyrosine-kinase pathway [J]. J Physiol Biochem. 2001, 57(1):15
[10] Lambard S,Galeraud-Denis I,Bouraima H,el a1.Expression of aromatase in human ejaculated spermatozoa:a putative marker of motility[J].Mol Hum Reprod.2003,9(3):117-24
[11] Arlt, Wiebke。Dehydroepiandrosterone replacement therapy[J]。Endocrinology and Diabetes.2006.13(3): 291-305
[12]Mitwally MF, Biljan MM, Casper RF.Pregnancy outcome after the use of an aromatase inhibitor for ovarian stimulation[J].Am J Obstet Gynecol.2005,192:381-386
[13]Stocco C, Kwintkiewicz J, Cai Z 。Identification of regulatory elements in the Cyp19 proximal promoter in rat luteal cells. [J]. Mol Endocrinol. 2007 , 39(4):211-221
[14]Kafali H, Iriadam M, Ozardl I, et al。Letrozole-Induced Polycystic Ovaries in the Rat: A New Model for Cystic Ovarian Disease [J]. Arch Med Res ,2004,35(2):103-108
[15]Deshpande RR, Chang MY, Chapman JC, et al. Alteration of cytokine production in follicular cystic ocaries induced in mice by neonatal estradiol injection [J]. Am J Reprod Immunol.2000, 44(2):80-88
[16]Auvray P, Nativelle C, Bureau R, et al. Study of substrate specificity of human aromatase by site directed mutagenesis [J].Eur J Biochem.2002, 269(5): 1393-1405
[17]Franks S, Mason H, White D, et al. Etiology of anovulation in polycystic ovary syndrome [J].steroids.1998, 63:306~307
[18]Ia Marca A, Morgante G, Palumbo M, et al。Insulin-lowering treatment reduces aromatase activity in response to follicle-stimulating hormone in women with polycystic ovary syndrome [J].Fertil Steril. 2002, 78(6):1234-1239
[19]Gerardo Barroso MD, Gerardo Menocal MD, et al. Comparison of the efficacy of the aromatase inhibitor letrozole and clomiphene citrate as adjuvants to recombinant follicle-stimulating hormone in controlled ovarian hyperstimulation: a prospective, randomized, blinded clinical trial [J]. Fertil Steril.2006, 86(5):1428~1431
[20]Catherine Hughes, Mustafa Elgasim. Genomic and post-genomic approaches to polycystic ovary syndrome—progress so far: Mini Review [J]. Human Reproduction.2006, 21(11):2766-2775