双氢睾酮诱导大鼠心肌细胞肥大模型的建立

应用胰蛋白酶分次消化法分离乳鼠心肌细胞,以差速贴壁法纯化心肌细胞,α-sarconme-actin抗体免疫细胞化学染色鉴定心肌细胞。心肌细胞在无血清无酚红培养基中培养48h后,用双氢睾酮(DHT)诱导心肌细胞肥大,建立心肌细胞肥大模型。24h后检测心肌细胞肥大的指标心肌细胞表面积;BCA法测定心肌细胞蛋白含量;半定量RTPCR两步法检测心肌细胞肥大的特征性基因—心房利钠因子(atrial natriuretic factor,ANF,β-肌球蛋白重链(β-myosin heavy chain,β-MHC)mRNA的表达。结果显示免疫细胞化学染色显示培养的心肌细胞纯度达到90%以上,心肌细胞分离良好。与对照组相比,10-8 mol/L的DHT能显著的增加心肌细胞表面积、蛋白质含量、ANP和β-MHC基因表达的增加(P0.01),心肌细胞肥大模型建立成功。     

双氢睾酮通过调控孕酮、雌二醇和细胞凋亡参与绵羊子宫功能

旨在探究双氢睾酮（dihydrotestosterone,DHT）是否通过调节孕酮（progesterone,P₄）、雌二醇（oestrogen,E₂）和细胞凋亡参与影响绵羊子宫功能,以揭示其在绵羊生殖生理中的潜在作用。本试验以1.5岁左右的雌性小尾寒羊为试验动物,检测卵泡期、黄体期和妊娠期子宫中DHT合成酶和雄激素受体（androgen receptor,AR）的表达变化。随后,体外培养绵羊子宫内膜上皮细胞,并用DHT （10^-10~10^-7 mol·L^-1）和AR拮抗剂氟他胺（Flu,10^-8 mol·L^-1）处理（n=3）。通过酶联免疫吸附试验、细胞免疫荧光、蛋白质印迹法和实时荧光定量PCR检测P₄和E₂水平、合成酶和受体表达。此外,还检测经DHT和Flu处理后,绵羊子宫内膜上皮细胞中凋亡因子Bcl-2相关X蛋白（Bcl-2-associated X protein,Bax）、B细胞淋巴瘤蛋白2（B cell lymphoma protein 2,Bcl-2）、半胱天冬酶3（caspase 3,CASP3）和活化半胱天冬酶3（active-caspase 3,Act-CASP3）的表达变化。结果表明,绵羊子宫不同时期DHT的合成和AR的表达存在差异,妊娠期子宫DHT合成及AR表达显著低于卵泡期和黄体期（P<0.05）。10^-10~10^-7 mol·L^-1DHT处理后,P₄合成酶表达显著上调（P<0.05）,在10^-8~10^-7 mol·L^-1 DHT时P₄合成显著增加（P<0.05）,在10^-10~10^-8 mol·L^-1 DHT时孕酮受体蛋白表达显著增加（P<0.05）,但10^-7mol·L^-1 DHT时孕酮受体蛋白表达显著下降（P<0.05）;在10^-8~10^-7 mol·L^-1 DHT时E₂相关合成酶显著减少,并且E₂水平显著下降（P<0.05）,在10^-9和10^-7 mol L^-1 DHT时E₂受体ERα蛋白显著下调（P<0.05）,在10^-10~10^-7 mol·L^-1 DHT时ERβ和GPER显著增加（P<0.05）。经Flu处理后部分解除DHT对P₄和E₂的调控。此外,10^-10~10^-7 mol·L^-1 DHT显著促进子宫内膜上皮细胞凋亡（P<0.05）。本研究证实DHT至少部分通过AR调节P₄和E₂合成及受体表达,影响细胞凋亡,参与调节子宫功能,这为进一步阐明雄激素参与调节子宫功能提供了新的基础和相关数据。     

Effect of dihydrotestosterone on mouse embryonic stem cells exposed to H2O2-induced oxidative stress

Oxidative stresses induced by reactive oxygen species (ROS) have been shown to be involved in several physiological and pathophysiological processes, such as cell proliferation and differentiation. Steroid hormones can protect cells against apoptosis or induce cell proliferation by several mechanisms. Among androgenic hormones, dihydrotestosterone (DHT) is generated by a 5α-reduction of testosterone. Unlike testosterone, DHT cannot be aromatized to estradiol, therefore DHT is considered a pure androgenic steroid. This study was conducted to examine the effect of DHT (10^-7 M) on H₂O₂ (10^-3 M) -induced injuries in mouse embryonic stem (ES) cells. H₂O₂ induced ROS generation and increased lipid peroxide formation and DNA fragmentation. These effects of H₂O₂ were inhibited by pretreatment with DHT. H₂O₂ also increased the phosphorylation of p38 MAPK, SAPK/JNK and nuclear factor kappa B (NF-κB), but DHT blocked these effects. Moreover, H₂O₂ decreased DNA synthesis and the levels of cell cycle regulatory proteins [cyclin D1, cyclin E, cyclin-dependent kinase (CDK) 2, and CDK 4]. These effects of H₂O₂ were inhibited by pretreatment with DHT. In conclusion, DHT may partially prevent H₂O₂-induced cell injury through inhibition of ROS and ROS-induced activation of p38 MAPK, SAPK/JNK and NF-κB in mouse ES cells.     

Follicle-stimulating hormone and luteinizing hormone regulate the synthesis mechanism of dihydrotestosterone in sheep granulosa cells

Steroid hormones and receptors play important roles in female reproduction, and their expression patterns affect follicular growth and development. To examine the expression of dihydrotestosterone (DHT) synthases (5α-reductases (5α-red1 and 5α-red2)) and androgen receptor (AR) during follicular development, and the regulation of DHT signalling by follicle-stimulating hormone (FSH) and luteinizing hormone (LH), we have used enzyme-linked immunosorbent assays, quantitative real-time polymerase chain reaction, immunohistochemical staining and Western blotting to examine DHT synthesis in small (≤2 mm), medium (2–5 mm) and large (≥5 mm) sheep follicles. Expression of 5α-red1, 5α-red2 and AR was observed in ovine ovaries, and with the development of follicles, the expressions of 5α-red1 and 5α-red2 mRNA and protein increased, but the levels of AR mRNA, protein and DHT level decreased. In addition, granulosa cells were treated with FSH (0.01, 0.1 and 1 international unit (IU)/ml), LH (0.01, 0.1 and 1 IU/ml) and testosterone (T, 10^–7 M) to evaluate the effects of FSH and LH on DHT and oestradiol (E2) synthesis and 5α-red1, 5α-red2 and AR expression. We found that FSH and LH upregulated 5α-red1 and 5α-red2 in sheep granulosa cells, but downregulated the concentration of DHT and expression of AR. Meanwhile, FSH and LH significantly upregulated the expression of aromatase (P450arom) and secretion of E2. This result indicates that although FSH and LH promote the expression of 5α-red1 and 5α-red2, T is not transformed into DHT, but E2. This study reveals the reason why DHT concentration is downregulated in large follicles and lays a foundation for further exploring the synthesis mechanism of DHT during follicular development.     

双氢睾酮对小鼠卵泡颗粒细胞增殖与抗苗勒管激素表达的影响

【目的】探讨双氢睾酮(DHT)对小鼠颗粒细胞增殖与抗苗勒管激素(AMH)表达的影响。【方法】给3周龄的昆明小鼠注射孕马血清促性腺激素(PMSG,10 IU/只)以获取颗粒细胞,颗粒细胞传代后48 h HE染色鉴定形态,绘制颗粒细胞的生长曲线,免疫荧光法鉴定促卵泡素受体(FSHR)的表达。当第2代颗粒细胞汇合度达到50%时,先用无血清的DMEM/F12培养基饥饿处理12 h,然后在培养基中添加不同浓度的DHT (0、10^-9、10^-8、10^-7、10^-6、10^-5 mol/L),培养48 h后检测颗粒细胞增殖情况,实时荧光定量PCR法及ELISA法分别测定AMH基因及蛋白的表达。在颗粒细胞培养液中分别添加10^-6 mol/L Flutamide (雄激素受体(AR)特异性抑制剂)、10^-7 mol/L DHT、10^-7 mol/L DHT+10^-6 mol/L Flutamide、10^-5 mol/L DHT、10^-5mol/L DHT+10^-8 mol/L 11-ketodihydrotestosterone (AR特异性激动剂),分别记为F、D7、DF、D5、DK组,以不添加药物为对照组。培养48 h后,检测各组颗粒细胞增殖及AMH蛋白含量。【结果】体外培养的小鼠颗粒细胞呈梭形或铺路石状,生长曲线呈S形,细胞普遍表达FSHR;与0 mol/L DHT组相比,10^-8、10^-7 mol/L DHT组细胞增殖分别显著和极显著增加(P＜0.05;P＜0.01),10^-5 mol/L DHT组细胞增殖显著降低(P＜0.05);10^-7 mol/L DHT组AMH基因的相对表达量和AMH蛋白含量均极显著增加(P＜0.01)。与D7组相比,DF组颗粒细胞增殖和AMH蛋白含量均极显著降低(P＜0.01);与D5组相比,DK组颗粒细胞增殖和AMH蛋白含量均极显著升高(P＜0.01)。【结论】10^-7 mol/L DHT能够显著促进颗粒细胞增殖和AMH表达,而10^-5 mol/L显著降低了颗粒细胞增殖以及AMH表达,且AR介导了DHT调控颗粒细胞增殖和AMH表达。