基于大鼠POI模型对减味寿胎丸雌激素样作用的研究

发布时间：2018-01-26 23:57

本文关键词： 减味寿胎丸卵巢功能不全雌激素样作用卵巢子宫下丘脑垂体 MCF-7细胞 ER荧光素酶报告基因　出处：《广州中医药大学》2016年博士论文　论文类型：学位论文

【摘要】：目的：探究减味寿胎丸对大鼠原发性卵巢功能不全(primary ovarian insufficiency, POI)模型的影响。探究减味寿胎丸提取物对MCF-7细胞增殖活力的影响。探究减味寿胎丸提取物对雌激素受体(Esterogen receptor, ER)荧光素酶报告基因表达的影响。方法：1.雷公藤多苷造大鼠POI模型SD大鼠分为5组：对照组、模型组、阳性对照组及减味寿胎丸高、中、低剂量组。雷公藤多苷片50mg/kg灌胃30d造大鼠POI模型,计算动情周期紊乱次数、卵巢及子宫指数。放射免疫检测血清雌二醇(Estradiol, E2)、卵泡刺激素(Follicle-stimulating hormone, FSH)、黄体生成素(Luteinizing hormone,LH)、 FSH/LH水平,HE染色法检测卵巢及子宫病理形态,western blot检测卵巢、子宫ER表达,免疫组化检测下丘脑及垂体ER α的表达。2.MCF-7细胞增殖实验MCF-7细胞96孔板接种,分为5组：1‰DMSO组、减味寿胎丸0.1mg/ml、0.01mg/ml、 0.001mg/ml组、0.1uM E2组,相应药物干预48h后以MTT法检测MCF-7细胞的增殖活力。3.ER荧光素酶报告基因检测实验MCF-7细胞接种6孔板,分为6组：1‰DMSO组、减味寿胎丸0.1mg/ml、0.01mg/ml、 0.001lmg/ml组、0.1uM E2及luM ICI 182,780组,将质粒用转入MCF-7细胞内,检测荧光素酶活性。结果：1.减味寿胎丸对大鼠POI模型的影响(1)动情周期：与空白组相比,模型组大鼠D1-D30、D21-D30动情周期紊乱次数较高,P0.01；与模型组相比,阳性组及减味寿胎丸高、中、低剂量组大鼠D1-D30、D21-D30动情周期紊乱次数较低,P0.5,差异有统计学意义。(2)血清激素水平：与空白组相比,模型组及减味寿胎丸低剂量组大鼠血清FSH较高,P0.001,有非常显著差异。与模型组相比,阳性组及减味寿胎丸高、中剂量组大鼠血清FSH较低,P0.05,差异有统计学意义。与空白组相比,模型组以及减味寿胎丸低剂量大鼠血清E2水平较低,P0.05,差异有统计学意义；与模型组相比,阳性组及减味寿胎丸高剂量组大鼠血清E2水平较高,P0.05,差异有统计学意义。与空白组相比,模型组及减味寿胎丸低剂量组大鼠血清FSH/LH较高,P0.05,差异有统计学意义；与模型组相比阳性组及减味寿胎丸高、中剂量组大鼠血清FSH/LH较低,P0.001,有非常显著差异。各组与空白组相比,P0.05,各组血清LH无明显差异；各组与模型组,P0.05,差异无统计学意义。(3)卵巢及子宫指数：与空白组相比,模型组卵巢指数较低,P0.001,有非常显著差异；与模型组相比,阳性组及减味寿胎丸高、中、低剂量组卵巢指数较高,P0.001,有非常显著差异。与空白组相比,模型组子宫指数较低,P0.001,有非常显著差异；与模型组相比,阳性组及减味寿胎丸高、中、低剂量组子宫指数较高,P0.01,差异有统计学意义。(4)卵巢及子宫病理形态：模型组卵泡较少,而其它组均可见各级卵泡。各组大鼠子宫病理形态无明显差异。(5)卵巢ER表达：与空白组相比,模型组卵巢ER α表达显著下调,P0.001,有非常显著差异；与空白组相比,阳性组及减味寿胎丸高、中、低剂量组大鼠卵巢ER α表达相比明显增高,P0.001,有非常显著差异。与空白组相比,模型组大鼠卵巢ERβ表达明显下调,P0.001,有非常显著差异；与模型组相比,阳性组及减味寿胎丸各剂量组大鼠卵巢ER β表达明显上调,P0.001,有非常显著差异。(6)子宫ER表达：与空白组相比,模型组及减味寿胎丸中、低剂量组大鼠子宫ER α表达明显下调,P0.001,有非常显著差异；与模型组相比,阳性组及减味寿胎丸各剂量组大鼠子宫ER α表达明显上调,P0.001,有非常显著差异。与空白组相比,模型组大鼠子宫ERβ表达明显下调,P0.001,有非常显著差异；与模型组相比,阳性组及减味寿胎丸高、中、低剂量组大鼠子宫ERβ表达明显上调,P0.001,有非常显著差异。(7)下丘脑及垂体ER α表达：与空白组相比,模型组大鼠下丘脑ERα表达下调,P0.01,有显著差异；同模型组相比,阳性组及减味寿胎丸高、中剂量组大鼠的下丘脑ER α表达明显增高,P0.01,有显著差异。与空白组相比,模型组大鼠的垂体ER α表达水平明显降低,P0.001,有非常显著差异；与模型组相比阳性组及减味寿胎丸高、中、低剂量组大鼠垂体ER α表达水平上调,P0.05,差异有统计学意义。2.减味寿胎丸对MCF-7细胞增殖活力的影响(1)与空白组相比0.001mg/ml减味寿胎丸提取物组及0.1uM E2组细胞增殖活力较高,P0.05,差异有统计学意义。(2)与空白组相比0.001mg/ml减味寿胎丸提取物组及0.1uM E2组细胞ER α表达水平较高,P0.05,差异有统计学意义。3.减味寿胎丸对ER荧光素酶报告基因表达的影响与空白组相比,0.1mg/ml、0.01mg/ml减味寿胎丸及1uM ICI 182,780组提取物组荧光素酶活性降低,P0.05,差异有统计学意义。0.001mg/ml减味寿胎丸提取物及0.1uM E2组荧光素酶活性提高,P0.05,差异有统计学意义。四、结论1.以减味寿胎丸干预雷公藤多苷POI大鼠模型,具有上调血清E2水平、下调血清FSH及FSH/LH水平、提高POI大鼠卵巢、子宫指数及卵泡数量,上调POI大鼠卵巢、子宫、下丘脑及垂体的ER表达,减少动情周期紊乱次数等作用。减味寿胎丸对POI大鼠的影响表现出了一定的雌激素样作用。2.0.001mg/ml减味寿胎丸提取物在体外表现出了一定的雌激素样作用,可通过上调ER α的表达提高MCF-7细胞的体外增殖活力。3.0.001mg/ml减味寿胎丸提取物在体外表现出了一定的雌激素样作用,可激活ER,提高ER荧光素酶报告基因的表达。0.1mg/ml及0.01mg/ml减味寿胎丸提取物可抑制ER活性,降低荧光素酶报告基因的表达。
[Abstract]:Objective : To investigate the effect of life - reducing Shoutai Pill on the expression of estrogen receptor ( ER ) luciferase reporter gene in rats . Compared with the model group , the serum levels of FSH / LH were lower in the model group and the lower - dose group than those in the model group ( P0.05 ) . Compared with the blank group , the serum levels of FSH / LH were lower in the model group and the life - reducing Shoutai pill group than those in the blank group . ( 3 ) The ovary and uterus index : Compared with the blank group , the ovary index of the model group was lower , P0.001 , there was very significant difference . Compared with the model group , there was a significant difference between the positive group and the life - reducing life - prolonging pill . Compared with the model group , the uterus index of the model group was lower , P0.01 , the difference was significant . Compared with the blank group , the expression of ER尾 in the uterus of the model group was significantly lower than that in the blank group ( P < 0.001 ) . Compared with the blank group , the expression of ER 尾 in the uterus of the model group was significantly lower than that in the blank group . ( 1 ) Compared with the blank group , the expression level of ER伪 in the hypothalamus of the model group was significantly lower than that in the blank group ( P < 0.01 ) . Compared with the blank group , the expression of ER 伪 in the hypothalamus of the model group was significantly lower than that in the blank group . ( 2 ) Compared with blank group , the expression level of ER伪 was higher than that in the blank group , the expression level of ER伪 was higher than that in the blank group , and the activity of luciferase reporter gene was decreased in the group of 0.01 mg / ml , 0.01 mg / ml and 0.1 uM E2 group .

【学位授予单位】：广州中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R285.5;R-332

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