孕期高雄激素对子代大鼠卵巢颗粒细胞P450芳香化酶表达的影响

目的探讨大鼠孕晚期暴露于高雄激素环境对子代雌鼠卵巢颗粒细胞P450芳香化酶(P450arom)表达的影响。方法选用性成熟雌性Wistar大鼠,随机分成实验组和对照组,按照1∶1比例与雄性Wistar大鼠合笼交配。在雌鼠妊娠第15～20天,实验组大鼠颈部皮下注射丙酸睾丸酮0.6 mg/d,对照组注射中性茶油0.6 mg/d。待其子代雌鼠2月龄时取双侧卵巢组织,应用半定量逆转录-聚合酶链反应(RT-PCR)检测实验组与对照组子代雌鼠P450aroma mRNA的表达;采用免疫组化(PV-6002)二步法测定实验组与对照组子代雌鼠P450aroma的蛋白表达。结果免疫组化法测得P450aroma蛋白在窦状卵泡颗粒细胞、黄体和卵巢间质中均有表达,且实验组P450aroma蛋白表达显著低于对照组(P<0.05)。RT-PCR法测得实验组与对照组卵泡颗粒细胞中P450aroma mRNA的相对表达量分别为(0.217 7±0.008 3)和(0.413 2±0.010 3),实验组显著低于对照组(P<0.05)。结论大鼠孕晚期暴露于高雄激素环境可以导致其子代雌鼠P450aroma表达下降,提示胎儿期暴露于高雄激素环境可能是导致PCOS的早期病因之一。     

血管内皮生长因子在卵巢过度刺激综合征发病机理中的作用

目的探讨血管内皮生长因子(VEGF)在卵巢过度刺激综合征(OHSS)大鼠模型发病机理中的作用。方法OHSS组、控制性超排卵(COH)组和对照组每组6只未成年雌性大鼠。采用酶联免疫吸附试验方法测定大鼠血清和腹腔冲洗液VEGF水平;免疫组织化学方法和逆转录聚合酶链反应技术检测卵巢组织VEGF蛋白及其mRNA的表达。结果OHSS组、COH组和对照组大鼠的血清VEGF水平分别为(76.17±18.19)、(50.68±12.83)和(53.68±13.09)ng/L;其中,OHSS组的VEGF水平显著高于COH组和对照组(P<0.05),而COH组和对照组比较无显著性差异。OHSS组、COH组和对照组大鼠腹腔冲洗液VEGF水平分别为(17.12±1.71)(、9.38±5.88)和(6.68±1.86)ng/L;其中,OHSS组的VEGF水平显著高于COH组和对照组(P<0.05),而COH组和对照组比较无显著性差异(P>0.05)。OHSS组大鼠卵巢组织VEGF蛋白表达的平均灰度(97.23±7.26)显著高于对照组(78.55±8.48)和COH组(87.35±7.32)(P<0.05);COH组与对照组比较无显著性差异。OHSS组大鼠卵巢组织VEGF mRNA表达的灰度比(1.23±0.23)显著高于对照组(0.68±0.13)和COH组(0.92±0.07)(P<0.01),COH组也显著高于对照组(P<0.05)。结论VEGF在OHSS发病过程中发挥作用。     

实验性多囊卵巢综合征大鼠脂肪组织中抵抗素基因的表达及其意义

目的研究脱氢表雄酮(DHEA)诱导SD幼年雌性大鼠多囊卵巢综合征(PCOS)动物模型的脂肪组织中抵抗素(resistin)mRNA的表达与其性激素变化及胰岛素抵抗(IR)的关系。方法以DHEA皮下注射21d龄SD雌性大鼠,观察卵巢重量及光镜(HE染色)、透视电镜下形态学改变,测定糖耐量、血清胰岛素、雌二醇(E2)、睾酮(T)、孕酮(P)、泌乳素(PRL)水平,采用逆转录聚合酶链反应(RT PCR)法检测脂肪组织resistinmRNA的表达。结果实验组卵巢重量显著高于对照组(P<0.05),实验组卵巢呈多囊样改变而黄体形成比例减少;实验组血清T、E2和空腹血糖、胰岛素水平明显高于对照组(分别为P<0.001、<0.05、<0.001、<0.05),空腹血胰岛素与空腹血糖乘积的倒数(1/FINS×FGC)显著高于对照组(P<0.05);PCOS大鼠白色脂肪组织resistinmRNA表达显著高于对照组(P<0.05)。结论DHEA诱导的PCOS大鼠动物模型与PCOS患者相似,伴有IR现象;由白色脂肪组织分泌的resistin在PCOS发生机制中起部分调节的作用,并使PCOS的IR进一步加重。     

高雄激素PCOS动物模型中抗苗勒管激素Ⅱ型受体蛋白及其基因甲基化分析

目的通过建立高雄激素诱导的多囊卵巢综合征(PCOS)大鼠模型,探讨抗苗勒管激素Ⅱ型受体(AMHRⅡ)在卵巢、子宫内膜中的表达及其基因启动子区域甲基化与PCOS发病及局部病理变化的关系。方法 45只雌性SD大鼠随机分两组:PCOS组皮下注射脱氢表雄酮(DHEA)建立PCOS模型,对照组同期皮下注射等量生理盐水;测定血清激素水平,结合卵巢组织HE染色切片结构改变评估PCOS模型;免疫组化法检测卵巢、子宫内膜组织AMHRⅡ蛋白的表达与定位;通过甲基化特异性PCR(MSP)定性分析两组大鼠血液、卵巢组织AMHRⅡ基因启动子区域的甲基化状态。结果血清性激素水平及卵巢切片结果均提示成功建立PCOS动物模型;免疫组化检测显示AMHRⅡ蛋白定位表达于细胞膜,在卵巢间质及卵泡颗粒细胞中均存在阳性表达,且在模型组中的表达强于对照组(P0.05);AMHRⅡ在子宫内膜中也存在表达,两组之间表达无统计学差异(P0.05);MSP结果显示两组中血液及卵巢组织所有DNA样本均检出部分甲基化条带,两组之间无统计学差异(P0.05)。结论 AMHRⅡ可能参与了PCOS中AMH调控卵泡发育的过程,但与子宫内膜病变无明显相关性;AMHRⅡ基因在启动子区域存在着部分甲基化,但这种甲基化状态与PCOS发病及局部组织病理变化无明显相关。     

多囊卵巢综合征中卵巢初级小卵泡bcl-2、bax的表达

目的 :探讨凋亡调节蛋白 bcl-2及 bax在多囊卵巢综合征 ( PCOS)患者的卵泡选择、发育的作用。方法 :1 8例 PCOS患者及 1 3名正常人 2 40个小卵泡按直径分为 4个组 ,采用免疫组织化学方法及 Konton Ibas灰度值的定量测定 ,用 SPSS软件统计 ,定量分析凋亡调节蛋白 bcl-2及 bax在 PCOS各级初级小卵泡中的表达。结果 :PCOS组各级窦前卵泡组间 bax蛋白表达有显著差异 ,随着卵泡直径的增加 bax表达明显增多 ,但直径 >1 2 0 μm的卵泡 bax表达减少 ,bcl-2 / bax灰度比值显著变小。 bcl-2蛋白表达相对稳定 :PCOS组直径在 60～ 1 2 0μm的卵泡 bcl-2表达显著高于正常对照组 ( P<0 .0 5 )。结论 :正常人及 PCOS卵巢组织各级窦前卵泡和窦卵泡均表达 bcl-2及 bax,随着卵泡的增大 ,bcl-2的表达增加 ,当卵泡直径 >1 2 0 μm时 ,细胞凋亡减弱 ,卵泡的增长加速。PCOS患者卵巢中各级窦前卵泡和窦卵泡凋亡蛋白 bcl-2及 bax的表达是正常的。     

瘦素与多囊卵巢综合征发病及体外受精结局的相关研究

目的探讨血清及卵泡液瘦素(leptin)水平与多囊卵巢综合征(PCOS)发病的关系和对体外受精-胚胎移植(IVF-ET)结局的影响。方法采用放射免疫法测定进行IVF-ET的20例PCOS患者(PCOS组)和20例非PCOS患者(对照组)卵泡液中leptin、胰岛素(INS)水平以及同期血清中leptin水平,PCOS患者经二甲双胍和口服避孕药(OC)预治疗三个月。结果PCOS组卵泡液及血清leptin水平均高于对照组(12.87±7.34 vs 10.10±5.15)、(13.65±7.75 vs 10.53±4.65),但差异无显著性,卵泡液及血清leptin水平均与卵泡液INS水平呈正相关(r=0.578、r=0.682,P均<0.01)。PCOS患者卵泡液低leptin组成熟卵细胞率高于高leptin组,差异有统计学意义(94%vs 86%,P<0.05),血清leptin水平与卵裂率呈负相关(r=-0.501,P<0.05)。结论PCOS患者体内leptin可能与卵泡液INS共同参与PCOS发生发展,leptin可能主要通过影响卵母细胞成熟、受精卵分裂而影响妊娠成功率。     

溴氰菊酯对大鼠卵巢甾体激素合成的影响

目的探讨溴氰菊酯(Deltamethrin,DLT)对大鼠卵巢甾体激素合成的影响及相关的分子机制。方法不同剂量DLT暴露处理大鼠和原代颗粒细胞;取DLT暴露后的大鼠血清和培养上清液,电化学发光法测定雌二醇和孕酮的水平;Western Blot法检测DLT暴露后大鼠卵巢组织及原代培养颗粒细胞中甾体激素合成关键酶:甾体激素合成急性调节蛋白(StAR)、胆固醇侧链裂解酶(P450scc)、芳香化酶的表达量,同时检测组蛋白去乙酰化酶Sirt1的蛋白表达。结果体、内外实验中,DLT各处理组大鼠血清、原代颗粒细胞培养上清液中雌二醇水平均显著升高(体内实验P<0.05,体外实验P<0.001);体内实验中DLT各处理组大鼠血清孕酮水平均显著降低(P<0.05),而体外实验中DLT各处理组原代颗粒细胞培养上清液孕酮水平无显著变化(P>0.05);体、内外实验中DLT各处理组大鼠卵巢组织、原代培养颗粒细胞中StAR、P450scc、芳香化酶以及Sirt1的表达量均显著升高(P<0.01)。结论 DLT能影响大鼠卵巢雌二醇和孕酮的合成,其机制可能与其关键酶表达的变化有关。     

能量限制下SIRT1高表达对大鼠卵巢生殖寿命的影响

目的观察能量限制后成年大鼠卵巢SIRT1高表达对卵巢功能和寿命的影响。方法 24只10周龄雌性SD大鼠,随机分为35%能量限制组和对照组。对照组不给予特殊处理自由取食,能量限制组予对照组食量的65%,每日调整饲料量,每周称体重。HE染色卵巢组织形态学观察及卵泡分类计数,免疫组织化学法观察SIRT1蛋白定位,Western blot检测SIRT1蛋白表达。结果能量限制组大鼠摄入能量少,体重的增长速度要低于对照组(P0.05),腹部脂肪明显少于对照组(P0.01);卵巢外观形态与对照组无异,原始卵泡数明显多于对照组(P0.01),窦状卵泡少于对照组(P0.01);SIRT1蛋白表达定位于卵母细胞的胞核,弱表达于胞浆,免疫印迹检测显示能量限制诱导大鼠卵巢组织SIRT1高表达,灰度值扫描与对照组有显著性差异(P0.05)。结论内源性SIRT1高表达可能是抑制原始卵泡的转化和发育,能量限制诱导SIRT1高表达可能对于延长卵巢的生殖寿命及提高卵巢的储备有积极作用。     

颗粒蛋白前体在高脂饮食诱导的肥胖PCOS大鼠卵巢中的表达

目的研究颗粒蛋白前体(PGRN)在肥胖PCOS大鼠卵巢中的表达情况,初步探讨PGRN在PCOS发病中的作用。方法选择SPF级23日龄SD雌性大鼠38只,使用随机数字表随机分为3组:正常对照组(n=8)、PCOS组(DHEA组,n=15)及肥胖PCOS组(DHEA+HFD组,n=15)。采用脱氢表雄酮联合高脂饲料(DHEA+HFD)诱导肥胖PCOS大鼠。HE染色光镜下观察PCOS大鼠卵巢的病理结构改变。酶联免疫吸附法测定血清睾酮(T)、雌二醇(E2)及空腹血糖(FBG)、空腹胰岛素(FINS)含量等,比较各组间的水平差异。采用免疫组织化学法检测各组卵巢PGRN的表达水平及分布情况。结果造模结束时,DHEA+HFD组体重显著高于对照组(P<0.01)及DHEA组(P<0.05)。DHEA组及DHEA+HFD组的FINS、稳态模型评估的胰岛素抵抗指数(HOMA-IR)均显著高于对照组(P<0.05)。DHEA组及DHEA+HFD组的睾酮(T)、FSH水平均显著升高(P<0.01)。DHEA组及DHEA+HFD组卵巢组织光镜下均表现出典型的多囊样改变。免疫组化结果显示各组大鼠卵巢中均可见PGRN蛋白表达,主要分布于卵泡的颗粒细胞层及黄体;DHEA组PGRN表达水平显著高于对照组(P<0.01),DHEA+HFD组PGRN表达水平显著高于DHEA组及对照组(P<0.01)。结论 PGRN在卵巢颗粒细胞层有表达。其在PCOS大鼠和肥胖PCOS大鼠卵巢组织中的差异性表达,提示PGRN可能通过作用于局部卵泡微环境,参与PCOS肥胖及慢性代谢性炎症的发生。     

补肾方对自然衰老大鼠睾酮调节作用及机制研究

目的:探讨补肾方对自然衰老大鼠睾酮调节作用及机制,为临床治疗迟发性睾丸功能减退提供理论和实验依据。方法:将32只18月龄老年雄性SD大鼠随机分为4组,自然衰老模型组,补肾方低、中、高剂量组,每组8只;另选4月龄青年雄性SD大鼠8只作为正常对照。正常对照组、自然衰老模型组予生理盐水,补肾方低剂量、中剂量、高剂量组分别按生药量3.25、7.5、15.0 g/kg体重予中药复方连续灌胃,各组给药3周后处死。采用苏木精-伊红(HE)染色观察大鼠睾丸组织形态,放免法检测大鼠血清睾酮水平,RT-PCR法检测大鼠类固醇合成急性调节蛋白(StAR)、细胞色素胆固醇侧链裂解酶(P450 scc)、3β-羟类固醇脱氢酶Ⅰ(3β-HSDⅠ)mRNA的相对表达。结果:睾丸组织病理切片显示补肾方干预后大鼠睾丸间质细胞数目增多,补肾方低、中、高剂量组血清睾酮水平[(6.74±1.56)、(8.50±1.99)、(12.41±2.91)nmol/L]与自然衰老模型组[(3.48±0.75)nmol/L]比较显著提高(P<0.05),睾酮合成相关酶StAR、P450 scc、3β-HSDⅠmRNA相对表达(StAR:0.74±0.29、0.83±0.32、1.35±0.50;P450 scc:0.72±0.36、1.02±0.30、1.41±0.37;3β-HSDⅠ:0.58±0.14、0.72±0.07、0.85±0.18)与自然衰老模型组(StAR:0.44±0.09;P450 scc:0.33±0.05;3β-HSDⅠ:0.34±0.02)比较均提高,其中高剂量组StAR,中、高剂量组P450 scc、3β-HSDⅠ的表达与自然衰老模型组比较差异有显著性(P<0.05)。结论:改善睾丸组织衰老的病理状态,提高睾酮合成酶表达可能是补肾方调节自然衰老大鼠睾酮水平的作用机制。     

Sources of oestrogen in the testis and reproductive tract of the male

The cytochrome P450 aromatase (P450arom) is the terminal enzyme responsible for the irreversible transformation of androgens into oestrogens and is present in the endoplasmic reticulum of various tissues throughout at least the phylum of vertebrates. The CYP 19 gene is unique and its expression is regulated in a tissue and more precisely in a cell-specific fashion via the alternative use of several promoters located in the first exons. The P450arom has been immunolocalized in germ cells of the mouse, brown bear and rooster. According to age, aromatase activity has been measured in immature and mature rat Leydig cells as well as in Sertoli cells, whereas in the pig, ram and human aromatase is mainly present in Leydig cells. In the adult rat testis, four complementary approaches (RTPCR, in situ hybridization, immunocytochemistry and the tritiated water assay) demonstrate that not only somatic cells but also mature germ cells represent a source of oestrogen synthesis. Taking into account the widespread distribution of oestrogen receptors (ER alpha & ER beta) in testicular cells and the genital tract of the male on the one hand, and the cross-talk between sex steroids and growth factors, and between membrane receptors and nuclear receptors for steroids on the other hand, it is anticipated that understanding of the pathophysiological roles of these 'female' hormones in the male will advance understanding of the hormonal regulation of male reproductive function. One of the future goals is to define oestrogen-targeted genes in the male gonad and indeed, a lot of work is now focused on this specific area in order to clarify the role of oestrogens in the reproductive tract of the male as well as to elucidate the regulation of aromatase gene expression.     

The association of aromatase （CYP19） gene variants wil sperm concentration and motility

The irreversible transformation of androgens into oestrogens is catalysed by cytochrome P450 aromatase. In the present study, we explored the contribution of the (TTTA)(n) polymorphism in the aromatase gene (CYP19) to sperm concentration and motility. Ninety normozoospermic and 60 oligospermic men were examined during infertility examinations. DNA was extracted from spermatozoa, and the CYP19 (TTTA)(n) polymorphism was genotyped by PCR. Genotype analysis revealed six CYP19 (TTTA)(n) alleles with 7-12 repeats. The allelic distribution of the CYP19 (TTTA)(n) polymorphism differed between normozoospermic and oligospermic men (P<0.01). Oligospermic men less frequently had long CYP19 alleles than did normozoospermic men (25 and 37.8%, respectively; P<0.02). The higher frequency of short CYP19 alleles in oligospermic men compared to normozoospermic men (43.3 and 28.3%, respectively; P<0.01) was primarily due to the distribution of the CYP19 (TTTA)(7) allele. The CYP19 (TTTA)(7) allele was associated with lower sperm concentration in normozoospermic men (P<0.01) and in the total study population (P<0.01); it was also associated with lower sperm motility in normozoospermic men (P<0.05) and in the total study population (P<0.01). In conclusion, the CYP19 (TTTA)(7) allele probably impairs aromatase activity, which in turn alters aromatase and oestrogen levels in the testis, leading to decreased sperm concentration and motility. These findings support the significance of cytochrome P450 aromatase in human spermatogenesis and consequently in semen quality.     

卵巢颗粒细胞中胰岛素和雄激素对芳香化酶和5α-还原酶1影响的研究

目的研究卵巢颗粒细胞中胰岛素对睾酮激素转化过程的影响。方法选用人卵巢颗粒细胞瘤样细胞系(KGN)作为研究对象。应用RT-PCR分析KGN细胞在梯度睾酮(1.25、2.5、5、10、20ng/ml)及胰岛素(1、10、100ng/ml)处理4h后CYP19a1和5RD5A1mRNA水平;Western-blot检测高浓度睾酮(10、20ng/ml)及梯度胰岛素处理KGN细胞24h后芳香化酶和5α-还原酶1蛋白水平;同步收集上清,应用放射性免疫法及酶联免疫法检测培养液上清睾酮、雌二醇及双氢睾酮的浓度。结果胰岛素剂量依赖性地上调CYP19a1的表达和活性,增加睾酮向雌二醇的转化;同时抑制5RD5A1的表达和活性,抑制睾酮向双氢睾酮的转化;转化比例改变但消耗水平不变,导致睾酮的异常累积。结论高雄激素环境下,胰岛素可通过影响睾酮转化酶促使睾酮转化异常。本实验为高胰岛素及高雄激素水平在卵巢微环境相互作用加剧PCOS病程提出了可能的机制。     

Aromatase and oestrogens in human male germ cells

The mammalian testis serves two main functions: production of spermatozoa and synthesis of steroids, among them oestrogens are the end products obtained from the irreversible transformation of androgens by aromatase (P450arom). Up today P450arom has been demonstrated in male germ cells of all mammals so far studied (mice, rat, bank vole, bear, monkey). In man Leydig cells and immature germ cells as well as ejaculated spermatozoa express a biologically active aromatase. Moreover germ cells and spermatozoa contain oestrogen receptors (ER-alpha and ER-beta) and it is of note that a truncated form of ER-alpha is present in spermatozoa. These observations clearly suggest that oestrogens are likely concerned in various stages of male germ cell development.     

Downregulation of intestinal cytochrome p450 in chronic renal failure

Chronic renal failure (CRF) is associated with a decrease in intestinal drug metabolism. The mechanisms remain poorly understood, but one hypothesis involves a reduction in cytochrome P450 levels. This study aimed to investigate the effects of CRF on intestinal cytochrome P450. Two groups of rats were defined, i.e., rats with CRF (induced by 5/6 nephrectomy) and control pair-fed rats. Total cytochrome P450 levels and protein and mRNA expression of cytochrome P450 isoforms, as well as in vitro N-demethylation of erythromycin (a probe for CYP3A activity) and 7-ethoxyresorufin o-deethylase activity (a probe for CYP1A), were assessed in intestinal microsomes. Body weights were similar in the two groups. Creatinine clearance was reduced by 77% (P < 0.001) in CRF rats, compared with control pair-fed animals. Total intestinal cytochrome P450 activity was reduced by 32% (P < 0.001) in CRF rats. CYP1A1 and CYP3A2 protein expression was considerably reduced (>40%, P < 0.001) in rats with CRF. CYP2B1, CYP2C6, and CYP2C11 levels were the same in the two groups. RT-PCR assays revealed marked downregulation of CYP1A1 and CYP3A2 gene expression in CRF rats (P < 0.001). Although intestinal cytochrome P450 levels were reduced in CRF, induction by dexamethasone was present. N-Demethylation of erythromycin and 7-ethoxyresorufin o-deethylase activity were decreased by 25% (P < 0.05) in CRF rats, compared with control rats. In conclusion, CRF in rats is associated with decreases in intestinal cytochrome P450 activity (mainly CYP1A1 and CYP3A2) secondary to reduced gene expression.     

青春期乳房肥大症乳腺组织中芳香化酶P450 mRNA的表达

目的 探讨芳香化酶P450 mRNA在青春期乳房肥大症乳腺组织中的表达及意义.方法 应用RT-PCR的方法测定15例青春期乳房肥大症乳腺组织中芳香化酶P450 mRNA的表达情况,并与15例正常乳腺组织中的芳香化酶P450 mRNA进行对比.结果 芳香化酶P450 mRNA的阳性表达率在乳房肥大组与正常乳腺组间差异无统计学意义(P>0.05).芳香化酶P450 mRNA阳性表达的病例中,青春期乳房肥大症乳腺组织中的平均测定值为0.202±0.048,正常乳腺组织为0.159±0.068,两者比较差异有统计学意义(P<0.05).结论 青春期乳房肥大症的发生、发展与乳腺组织中芳香化酶P450 mRNA的表达情况有关.     

Downregulation of hepatic cytochrome P450 in chronic renal failure

Chronic renal failure (CRF) is associated with a decrease in drug metabolism. The mechanism remains poorly understood. The present study investigated the repercussions of CRF on liver cytochrome P450 (CYP450). Three groups of rats were defined: control, control paired-fed, and CRF. Total CYP450 activity, protein expression of several CYP450 isoforms as well as their mRNA, and the in vitro N-demethylation of erythromycin were assessed in liver microsomes. The regulation of liver CYP450 by dexamethasone and phenobarbital was assessed in CRF rats. Compared with control and control paired-fed rats, creatinine clearance was reduced by 60% (P: < 0.01) in CRF rats. Weight was reduced by 30% (P: < 0.01) in control paired-fed and CRF rats, compared with control animals. There was no difference in the CYP450 parameters between control and control paired-fed. Compared with control paired-fed rats, total CYP450 was reduced by 47% (P: < 0.001) in CRF rats. Protein expression of CYP2C11, CYP3A1, and CYP3A2 were considerably reduced (>40%, P: < 0.001) in rats with CRF. The levels of CYP1A2, CYP2C6, CYP2D, and CYP2E1 were the same in the three groups. Northern blot analysis revealed a marked downregulation in gene expression of CYP2C11, 3A1, and 3A2 in CRF rats. Although liver CYP450 was reduced in CRF, its induction by dexamethasone and phenobarbital was present. N-demethylation of erythromycin was decreased by 50% in CRF rats compared with control (P: < 0.001). In conclusion, CRF in rats is associated with a decrease in liver cytochrome P450 activity (mainly in CYP2C11, CYP3A1, and 3A2), secondary to reduced gene expression.