生理周期及致痫后雌性大鼠海马细胞色素P450芳香化酶的动态表达
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摘要
目的:(1)观察雌性大鼠不同生理周期细胞色素P450芳香化酶在海马神经元和星形胶质细胞的表达;(2)观察去势雌性大鼠致痫后不同时间点细胞色素P450芳香化酶在海马神经元和星形胶质细胞的表达变化,及其与癫痫发作严重程度的关系,了解内源性雌二醇的合成来源。
方法:(1)成年Sprague-Dawley(SD)大鼠(体重250±10g)分为六组:雌性大鼠动情前期组、动情期组、动情后期组、动情间期组、去势组及雄性大鼠组,每组6只,心脏灌流断头取脑后行冰冻切片;(2)将成年SD雌性大鼠(体重250±10g)行双侧卵巢切除术后饲养2周(目标体重290±10g),术后第15天随机分为12组,每组6只,其中6组为KA组,6组为NS对照组,制备杏仁核点燃模型,记录模型点燃的潜伏期、观察癫痫发作的频率及程度,各组分别在点燃成功后第1、2、3、4、5、6小时心脏灌流断头取脑后,行冰冻切片;(3)免疫荧光双标法检测细胞色素P450芳香化酶在海马神经元和星形胶质细胞的表达变化:a、NeuN、GFAP分别标记神经元和星形胶质细胞;b、Aromatase抗体标记芳香化酶c、荧光显微镜及激光共聚焦显微镜观察芳香化酶及其细胞定位;(4)图片行细胞计数进行定量分析。
结果:(1)免疫荧光结果显示:大鼠海马CA1-CA3区及DG区均可见到芳香化酶免疫阳性表达,其中CA1区、DG区表达较为明显,且细胞浆、细胞核均有表达,免疫荧光双标染色显示芳香化酶主要表达于神经元上;(2)动情期雌性大鼠海马各区免疫阳性细胞数均较其他各期增多,其中CA1区、DG区的差异有统计学意义(p＜0.05),去势后的雌性大鼠海马芳香化酶表达下降,与各生理周期相比均有统计学差异(p＜0.05);(3)雄性大鼠海马芳香化酶的表达高于雌性大鼠各生理周期,差异有统计学意义(p＜0.05);(4)致痫后1～6小时,海马芳香化酶的免疫阳性细胞仍以神经元表达为主,表达无明显变化趋势,差异没有统计学意义,与NS组比较差别也无统计学意义;(5)致痫后5～6小时,芳香化酶在星形胶质细胞上表达明显增多,主要见于CA1区,与之前0～4小时相比差异有统计学意义(p＜0.05),与NS组对应时间点相比,差异也有统计学意义(p＜0.05);(6)KA注射后20-30min出现轻度痫性发作,3小时发作程度最重,随后呈下降趋势,芳香化酶在星形胶质细胞上的表达与癫痫发作程度无相关性。性雌二醇以神经元合成为主;2)不同生理周期雌性大鼠海马芳香化酶表达存在动态变化,动情期表达最高,故考虑不同生理周期雌性大鼠海马内源性雌二醇的合成存在动态变化;3)大鼠海马芳香化酶的表达存在性别差异;4)癫痫发作后芳香化酶仍以神经元表达为主,星形胶质细胞可见少量表达,考虑癫痫发作后海马内源性雌二醇合成仍以神经元合成为主;5)致痫后,芳香化酶在神经元上的表达无明显变化,海马内源性雌二醇的变化可能与芳香化酶蛋白含量变化无关;6)在癫痫发作末期出现芳香化酶在星形胶质细胞上的表达明显增加,考虑星形胶质细胞合成雌二醇。
Objective
(1)To observe the expression of cytochrome P450 aromatase within neurons and astrocytes in the hippocampus of female rats with different estrous cycle.
(2)To observe the changes of P450arom expression within neurons and astrocytes in the hippocampus after the onset of seizures,define the relationship to the severity of seizures,and the source of brain endogenous estradiol.
Methods
(1)Adult Sprague-Dawley rats(weight240～260g) were divided into 6 groups: proestrus group,estrus group,metestrus group,diestrus group,ovariectomized group of female rats and male group,each group has 6 rats.After perfused with 4℃saline and 4%paraformaldehyde solution,the brains were harvested after decapitation. These brains were fixed with 4%paraformaldehyde solution for 12h and dehydrated with 10%,20%,30%saccharose gradient.Frozen sections were prepared for staining. (2)The female SD rats were raised for two weeks after ovariectomy (weight280～300g),and randomly divided into 12 groups,including 6 KA groups and 6 NS control groups.On the 15th day,the rats were anesthetized and fixed to the stereotaxic apparatus,1μg dose of KA were injected into the right amygdale slowly to induce the complex partial seizure(5μl NS was injected into the right amygdale of the NS group rats).The latency to severe seizure stage,the frequency and duration of the seizure were recorded.The samples were prepared according to the protocals described in part one after 1h,2h,3h,4h,5h,6h after seizure were collected respectively.(3)Dual immunofluorescence staining were used to measure the positive cells of aromatase in neurons and astrocytes:a.Neurons and astrocytes were marked with NeuN and GFAP antibodies.b.Aromatase was marked with aromatase polyclonal antibody.c.Fluorescence microscope and Laser confocal microscope were selected for the staining results.
Results
(1)The results of immunofluorescence showed the P450arom immunoreactive cells distributed in the CA1 to CA3 and DG region of hippocampus,maily in CA1 and DG region.They expressed on both membrane and nuclea of neuron.(2)A large quantity of P450arom immunoreactive cells were found in the estrus group.In CA1 and DG regions,there were statistically significant difference with other groups of female rats. The P450arom expression of ovariectomized group in hippocampus were obviously weaker than normal female rats(p＜0.05).(3)Statistically significant difference were also found between male group and female group,while male group had a strong expression.(4)The P450arom still maily expressed in neuron after seizure onset,and the quantity of immunoreactive cells didn't have a statistically significant change during the stage of 1-6 hours after seizure onset.(5)At the 5th and 6th hours after seizure onset,the expression of P450arom appeared on astrocyte.The the quantity of immunoreactive cells coexpression with GFAP had a statistically significant difference compared with the 0-4 hours after seizure onset and the NS groups of the same time point.(6)Approximately 20-30min after KA intra-amgydale injection,the rats began to show mild seizure.The peak were reached in 180 min after KA injection and then the seizure was alleviated.There were no correlation between seizure severity scale and quanitity of P450arom immunoreactive cells.
Conclusions
The P450arom expression could be detected in hippocampus and maily localized in neuron,which indicated estradiol can be synthetized locally in hippocampus of neurons.(2)There were dynamic changes of the quanitity of P450arom expression during estrous cycle.(3)There were sex difference of P450arom expression in rats.(4) The expression of P450arom were still maily localized in neuron after seizure onset, however there were a small quanitity of P450arom expression appeared on astrocyte. (5)The quantity of P450arom in neuron didn't have a significant change after seizure onset.(6)The quantity of P450arom in astrocyte obviously increased in telophase of epileptic paroxysm,which indicated estradiol can also be synthetized by astrocyte in seizure rats hippocampus.
方法:(1)成年Sprague-Dawley(SD)大鼠(体重250±10g)分为六组:雌性大鼠动情前期组、动情期组、动情后期组、动情间期组、去势组及雄性大鼠组,每组6只,心脏灌流断头取脑后行冰冻切片;(2)将成年SD雌性大鼠(体重250±10g)行双侧卵巢切除术后饲养2周(目标体重290±10g),术后第15天随机分为12组,每组6只,其中6组为KA组,6组为NS对照组,制备杏仁核点燃模型,记录模型点燃的潜伏期、观察癫痫发作的频率及程度,各组分别在点燃成功后第1、2、3、4、5、6小时心脏灌流断头取脑后,行冰冻切片;(3)免疫荧光双标法检测细胞色素P450芳香化酶在海马神经元和星形胶质细胞的表达变化:a、NeuN、GFAP分别标记神经元和星形胶质细胞;b、Aromatase抗体标记芳香化酶c、荧光显微镜及激光共聚焦显微镜观察芳香化酶及其细胞定位;(4)图片行细胞计数进行定量分析。
结果:(1)免疫荧光结果显示:大鼠海马CA1-CA3区及DG区均可见到芳香化酶免疫阳性表达,其中CA1区、DG区表达较为明显,且细胞浆、细胞核均有表达,免疫荧光双标染色显示芳香化酶主要表达于神经元上;(2)动情期雌性大鼠海马各区免疫阳性细胞数均较其他各期增多,其中CA1区、DG区的差异有统计学意义(p＜0.05),去势后的雌性大鼠海马芳香化酶表达下降,与各生理周期相比均有统计学差异(p＜0.05);(3)雄性大鼠海马芳香化酶的表达高于雌性大鼠各生理周期,差异有统计学意义(p＜0.05);(4)致痫后1～6小时,海马芳香化酶的免疫阳性细胞仍以神经元表达为主,表达无明显变化趋势,差异没有统计学意义,与NS组比较差别也无统计学意义;(5)致痫后5～6小时,芳香化酶在星形胶质细胞上表达明显增多,主要见于CA1区,与之前0～4小时相比差异有统计学意义(p＜0.05),与NS组对应时间点相比,差异也有统计学意义(p＜0.05);(6)KA注射后20-30min出现轻度痫性发作,3小时发作程度最重,随后呈下降趋势,芳香化酶在星形胶质细胞上的表达与癫痫发作程度无相关性。性雌二醇以神经元合成为主;2)不同生理周期雌性大鼠海马芳香化酶表达存在动态变化,动情期表达最高,故考虑不同生理周期雌性大鼠海马内源性雌二醇的合成存在动态变化;3)大鼠海马芳香化酶的表达存在性别差异;4)癫痫发作后芳香化酶仍以神经元表达为主,星形胶质细胞可见少量表达,考虑癫痫发作后海马内源性雌二醇合成仍以神经元合成为主;5)致痫后,芳香化酶在神经元上的表达无明显变化,海马内源性雌二醇的变化可能与芳香化酶蛋白含量变化无关;6)在癫痫发作末期出现芳香化酶在星形胶质细胞上的表达明显增加,考虑星形胶质细胞合成雌二醇。
Objective
(1)To observe the expression of cytochrome P450 aromatase within neurons and astrocytes in the hippocampus of female rats with different estrous cycle.
(2)To observe the changes of P450arom expression within neurons and astrocytes in the hippocampus after the onset of seizures,define the relationship to the severity of seizures,and the source of brain endogenous estradiol.
Methods
(1)Adult Sprague-Dawley rats(weight240～260g) were divided into 6 groups: proestrus group,estrus group,metestrus group,diestrus group,ovariectomized group of female rats and male group,each group has 6 rats.After perfused with 4℃saline and 4%paraformaldehyde solution,the brains were harvested after decapitation. These brains were fixed with 4%paraformaldehyde solution for 12h and dehydrated with 10%,20%,30%saccharose gradient.Frozen sections were prepared for staining. (2)The female SD rats were raised for two weeks after ovariectomy (weight280～300g),and randomly divided into 12 groups,including 6 KA groups and 6 NS control groups.On the 15th day,the rats were anesthetized and fixed to the stereotaxic apparatus,1μg dose of KA were injected into the right amygdale slowly to induce the complex partial seizure(5μl NS was injected into the right amygdale of the NS group rats).The latency to severe seizure stage,the frequency and duration of the seizure were recorded.The samples were prepared according to the protocals described in part one after 1h,2h,3h,4h,5h,6h after seizure were collected respectively.(3)Dual immunofluorescence staining were used to measure the positive cells of aromatase in neurons and astrocytes:a.Neurons and astrocytes were marked with NeuN and GFAP antibodies.b.Aromatase was marked with aromatase polyclonal antibody.c.Fluorescence microscope and Laser confocal microscope were selected for the staining results.
Results
(1)The results of immunofluorescence showed the P450arom immunoreactive cells distributed in the CA1 to CA3 and DG region of hippocampus,maily in CA1 and DG region.They expressed on both membrane and nuclea of neuron.(2)A large quantity of P450arom immunoreactive cells were found in the estrus group.In CA1 and DG regions,there were statistically significant difference with other groups of female rats. The P450arom expression of ovariectomized group in hippocampus were obviously weaker than normal female rats(p＜0.05).(3)Statistically significant difference were also found between male group and female group,while male group had a strong expression.(4)The P450arom still maily expressed in neuron after seizure onset,and the quantity of immunoreactive cells didn't have a statistically significant change during the stage of 1-6 hours after seizure onset.(5)At the 5th and 6th hours after seizure onset,the expression of P450arom appeared on astrocyte.The the quantity of immunoreactive cells coexpression with GFAP had a statistically significant difference compared with the 0-4 hours after seizure onset and the NS groups of the same time point.(6)Approximately 20-30min after KA intra-amgydale injection,the rats began to show mild seizure.The peak were reached in 180 min after KA injection and then the seizure was alleviated.There were no correlation between seizure severity scale and quanitity of P450arom immunoreactive cells.
Conclusions
The P450arom expression could be detected in hippocampus and maily localized in neuron,which indicated estradiol can be synthetized locally in hippocampus of neurons.(2)There were dynamic changes of the quanitity of P450arom expression during estrous cycle.(3)There were sex difference of P450arom expression in rats.(4) The expression of P450arom were still maily localized in neuron after seizure onset, however there were a small quanitity of P450arom expression appeared on astrocyte. (5)The quantity of P450arom in neuron didn't have a significant change after seizure onset.(6)The quantity of P450arom in astrocyte obviously increased in telophase of epileptic paroxysm,which indicated estradiol can also be synthetized by astrocyte in seizure rats hippocampus.
引文
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