二仙汤及其组方中药对幼年大鼠肾上腺雌激素受体表达的影响论文.docVIP

　　二仙汤及其组方中药对幼年大鼠肾上腺雌激素受体表达的影响论文 .freelechanism of Er-xian Decoction and its ponent herbs on the expression of extrogen receptor (ER) in adrenal development rats. Methods Fifty-four development female SD rats ly divided into nine groups：normal control group (A), positive control group (B), Er-xian Decoction group (C), Herba Epimedii group (D), Rhizoma Curculiginis group (E), Radix Morindae Officinalis group (F), Radix Angelicae Sinensis group (G), Cortex Phellodendri group (H), Rhizoma Anemarrhenae group (I). After the rats inistrated for six days, serum easure testosterone (T). Bilateral adrenal gland bedded by paraffin. Mean optical density (MOD) munohistochemical method and image analyzing system. Results The adrenal coefficient ainly observed in the cytoplasm of zona glomerulosa and zona fasciculate. MOD in group C ainly observed in nucleus of zona glomerulosa and zona fasciculate. MOD in group C, D, E and I ing Shen (Herba Epimedii, Rhizoma Curculiginis) and nourishing Yin (Rhizoma Anemarrhenae) play a phytoestrogen role by influencing the expression of ERα and ER β in the adrenal cortex. Key L药液;二仙汤按淫羊藿、仙茅、巴戟天∶当归、黄柏、知母为3∶2配制成原药材含量1 g/mL药液;阳性药物为己烯雌酚(批,0.5 mg×100片/瓶,合肥久联制药有限公司生产。以上药物均购自北京中医药大学东直门医院。 1.3 试剂 一抗：ERα(MC-20)和ERβ(H-150)为Santa Cruz公司产品;过氧化物酶标记的链霉卵白素染色试剂盒(SP-9001)购自北京中杉金桥生物技术有限公司;血清睾酮(T)检测试剂盒购自北京华英生物技术研究所。 2 实验方法 2.1 分组与给药 将实验动物按体重均衡和随机的原则分为9组：正常对照组、阳性对照组、二仙汤组以及6个单味药组。实验动物适应性饲养3 d后开始实验。正常对照组给予等体积蒸馏水;阳性对照组按0.18 mg/(kg·d)给予己烯雌酚;二仙汤组按7.5 g/(kg·d)给药;淫羊藿、仙茅、巴戟天组按1.5 g/(kg·d)给药;当归、黄柏和知母组按1.0 g/(kg·d)给药。灌胃分早、晚2次进行,给药均持续6 d。 2.2 动物取材与血清提取 给药6 d,处死前禁食12 h。用10%水合氯醛按4 mL/kg腹腔注射麻醉,沿大鼠腹正中线打开腹腔,自腹主动脉取血,经室温静置2 h后,3 000 r/min离心10 min,取其上清液,分别保存于EP管中,-20 ℃保存备用。将肾上腺与周围组织完全剥离后取出,称重,用中性甲醛固定后制成石蜡切片。 2.3 血清睾酮的测定 具体步骤参照所购试剂盒说明书。 2.4 免疫组织化学染色步骤 石蜡切片脱蜡至水,微波修复抗原10 min;入3%过氧化氢室温孵育30 min;正常山羊血清封闭液室温孵育20 min,倾去,分别用兔抗大鼠ERα或ERβ抗体(1∶150,Santa Cruz)孵育切片(4 ℃,过夜);羊抗兔二抗37 ℃孵育30 min,辣根酶标记链霉卵白素工作液37℃孵育30 min,DAB染色。每步骤间用0.O1 mol/L PBS充分洗涤,常规脱水,透明,封片,光镜下观察,摄片。染成棕黄色颗粒者为阳性。