雌激素的神经保护作用及其分子机制
南京大学医学院附属鼓楼医院神经内科 (210008)
【摘要】
目的：探讨雌激素的神经保护作用及其分子机制
方法：1. 雌激素受体α基因敲除小鼠作一侧大脑中动脉梗塞模型，野生型小鼠作为对照。2. 原代小鼠大脑皮质神经元培养，作缺氧模型，293细胞珠作信息通路研究，因其无雌激素受体α。3. 用不同剂量的雌激素处理缺血性小鼠和缺氧的神经元，即25μg/日(E-25)，50μg/日(E-50)，连续3 d，100μl的芝麻油作为对照(veh)。 4. TCC和PI / calcein 测定脑缺血的梗塞面积和缺氧后神经元损伤情况。5.克隆雌激素受体α、细胞内转染、基因报告系统、细胞免疫组化方法测定雌激素神经保护的信号通路。
结果：1.高剂量的雌激素(50 μg/d×3 d)明显减轻基因敲除型及野生型小鼠的脑梗塞面积，低剂量雌激素(25μg/d×3 d)只能减轻野生型小鼠的脑梗塞面积，野生型梗塞面积分别为59.6±4.10(veh), 26.3±3.9(E-25)，22.4±4.25(E-50)；基因敲除型：25.6±3.7(veh)，23.5±4.12(E-25)，8.35±3.47(E-50)。2.雌激素显著减轻缺氧性神经元损伤，PI/calcein 染色结果为神经细胞死亡率(%)分别为：37.07±3.60(veh), 27.5±3.04(E-5nM)，23.35±5.01(E-10nM)，18.82±3.31(E-15nm ),15.6±2.85(E-20nM)，25.05±6.28(E-50nM)。3.小鼠大脑皮质表达雌激素受体α，293细胞不表达，80%的受体表达在细胞浆和核内，20%为膜型的受体，用CAT和荧光素报告基因发现雌激素可激活核内转录水平的基因途径，即ERE通路(Estrogen Response Element)，将克隆的雌激素受体α转染进293细胞中，也可激活此途径.4. 细胞免疫组化结果示雌激素受体α与MAPK基因表达在同一部位，即膜型雌激素受体可激活非基因途径，MAPK途径，此途径反应较快，在几秒钟内即可反应，在急性脑血管病中的应激状态中非常有意义。 结论：雌激素明显减轻缺血，缺氧性神经元损害，通过结合其受体α而起作用，核型雌激素受体激活ERE转录水平的基因通路，膜型受体激活MAPK的非基因途径。
, 百拇医药
Neuroprotection of Estrogen and It’s Molecular Machanism
XU Yun, WANG Nan,WANG chong,Department of Neurology, The affiliated Drum Tower Hospital of Nanjing University, Medical School. Nanjing 210008
【Abstract】Objective：To study Neuroprotection of Estrogen and It’s Molecular Machanism. Methods 1.Estrogen receptor α(ERα)-knockout mice with MCAO, wild type mice as control. 2. Primary mice cortex neurons culture with OGD, 293 cell line which has no estrogen receptor expression for signal research. 3. Estrogen in different dose treated MCAO mice and OGD-neurons, that is 25ug/day(E-25), 50ug/day(E-50), three days and 100ul sesame oil as control. 4.Infact was detacted by TTC and neurons death determined by PI/Calcein. 5.Estrogen signals were studied by Estrogen receptors cloning, transfection, gene report assay and immunohistochemistry. Results：1.Higher dose estrogen(E-50) decrese ischemic infarct with knockout and wild type mice siganificatly. Lower dose only was usable for wild type. Ischemic infarct(%) were as following: wild type,59.6±4.10(veh), 26.3±3.9(E-25)，22.4±4.25(E-50)； knockout mice, 25.6±3.7(veh)，23.5±4.12(E-25)，8.35±3.47(E-50). 2. OGD-neurons death was decreased by Estrogen using PI/Calcein(%), that is 37.07±3.60 (veh), 27.5±3.04(E-5nM)，23.35±5.01(E-10nM)，18.82±3.31(E-15Nm ),15.6±2.85(E-20nM)，25.05±6.28(E-50nM)。 3. ERα expressed in neuron, not in 293 cell. 80% located in nuclear and 20% in neurites. Estrogen activates ERE function using CAT or Lucifarase assay. GFP- ERαfusions which transfected into 293 cells also have this function. 4.ERαexpressed on neurites and colacolizated with MAP Kinase protein using immunohistochemistry, that is membrane-ER activated rapid nongenomic signal pass way and it’s important in acute cerebral blood vessel diseas. Conclusion：Estrogen activated ER and protected brain and neurons from ischemic or hypoxia injury. ER in nuclear activated ERE genetic pass way and ER in membrane activated MAP Kinase rapid nongenetic pass way., 百拇医药(徐运 王岚 王翀 管得宁 黄嵘 王中原 朱琳 朱文彬 王晓云 倪秀石)
【摘要】
目的：探讨雌激素的神经保护作用及其分子机制
方法：1. 雌激素受体α基因敲除小鼠作一侧大脑中动脉梗塞模型，野生型小鼠作为对照。2. 原代小鼠大脑皮质神经元培养，作缺氧模型，293细胞珠作信息通路研究，因其无雌激素受体α。3. 用不同剂量的雌激素处理缺血性小鼠和缺氧的神经元，即25μg/日(E-25)，50μg/日(E-50)，连续3 d，100μl的芝麻油作为对照(veh)。 4. TCC和PI / calcein 测定脑缺血的梗塞面积和缺氧后神经元损伤情况。5.克隆雌激素受体α、细胞内转染、基因报告系统、细胞免疫组化方法测定雌激素神经保护的信号通路。
结果：1.高剂量的雌激素(50 μg/d×3 d)明显减轻基因敲除型及野生型小鼠的脑梗塞面积，低剂量雌激素(25μg/d×3 d)只能减轻野生型小鼠的脑梗塞面积，野生型梗塞面积分别为59.6±4.10(veh), 26.3±3.9(E-25)，22.4±4.25(E-50)；基因敲除型：25.6±3.7(veh)，23.5±4.12(E-25)，8.35±3.47(E-50)。2.雌激素显著减轻缺氧性神经元损伤，PI/calcein 染色结果为神经细胞死亡率(%)分别为：37.07±3.60(veh), 27.5±3.04(E-5nM)，23.35±5.01(E-10nM)，18.82±3.31(E-15nm ),15.6±2.85(E-20nM)，25.05±6.28(E-50nM)。3.小鼠大脑皮质表达雌激素受体α，293细胞不表达，80%的受体表达在细胞浆和核内，20%为膜型的受体，用CAT和荧光素报告基因发现雌激素可激活核内转录水平的基因途径，即ERE通路(Estrogen Response Element)，将克隆的雌激素受体α转染进293细胞中，也可激活此途径.4. 细胞免疫组化结果示雌激素受体α与MAPK基因表达在同一部位，即膜型雌激素受体可激活非基因途径，MAPK途径，此途径反应较快，在几秒钟内即可反应，在急性脑血管病中的应激状态中非常有意义。 结论：雌激素明显减轻缺血，缺氧性神经元损害，通过结合其受体α而起作用，核型雌激素受体激活ERE转录水平的基因通路，膜型受体激活MAPK的非基因途径。
, 百拇医药
Neuroprotection of Estrogen and It’s Molecular Machanism
XU Yun, WANG Nan,WANG chong,Department of Neurology, The affiliated Drum Tower Hospital of Nanjing University, Medical School. Nanjing 210008
【Abstract】Objective：To study Neuroprotection of Estrogen and It’s Molecular Machanism. Methods 1.Estrogen receptor α(ERα)-knockout mice with MCAO, wild type mice as control. 2. Primary mice cortex neurons culture with OGD, 293 cell line which has no estrogen receptor expression for signal research. 3. Estrogen in different dose treated MCAO mice and OGD-neurons, that is 25ug/day(E-25), 50ug/day(E-50), three days and 100ul sesame oil as control. 4.Infact was detacted by TTC and neurons death determined by PI/Calcein. 5.Estrogen signals were studied by Estrogen receptors cloning, transfection, gene report assay and immunohistochemistry. Results：1.Higher dose estrogen(E-50) decrese ischemic infarct with knockout and wild type mice siganificatly. Lower dose only was usable for wild type. Ischemic infarct(%) were as following: wild type,59.6±4.10(veh), 26.3±3.9(E-25)，22.4±4.25(E-50)； knockout mice, 25.6±3.7(veh)，23.5±4.12(E-25)，8.35±3.47(E-50). 2. OGD-neurons death was decreased by Estrogen using PI/Calcein(%), that is 37.07±3.60 (veh), 27.5±3.04(E-5nM)，23.35±5.01(E-10nM)，18.82±3.31(E-15Nm ),15.6±2.85(E-20nM)，25.05±6.28(E-50nM)。 3. ERα expressed in neuron, not in 293 cell. 80% located in nuclear and 20% in neurites. Estrogen activates ERE function using CAT or Lucifarase assay. GFP- ERαfusions which transfected into 293 cells also have this function. 4.ERαexpressed on neurites and colacolizated with MAP Kinase protein using immunohistochemistry, that is membrane-ER activated rapid nongenomic signal pass way and it’s important in acute cerebral blood vessel diseas. Conclusion：Estrogen activated ER and protected brain and neurons from ischemic or hypoxia injury. ER in nuclear activated ERE genetic pass way and ER in membrane activated MAP Kinase rapid nongenetic pass way., 百拇医药(徐运 王岚 王翀 管得宁 黄嵘 王中原 朱琳 朱文彬 王晓云 倪秀石)