雌鼠不同生殖周期特定脑区突触外GABAA受体表达研究
发布时间：2018-07-25 21:21
【摘要】：女性生殖周期雌孕激素呈现周期性变化，尤其是妊娠过程和分娩后激素的较大变化使得女性出现各种各样情绪障碍及各种临床症状。有研究认为,妇女的以上各类精神疾病均有女性生殖周期雌、孕激素等卵巢甾体激素的波动所引发。这些临床症状的出现与孕激素对GABAA调节系统的作用有关。但体内神经甾体激素及其中枢神经系统的调控变化与此类抑郁症相关性的认识尚未统一，现有的研究尚不足以揭示此类疾病发生的实质。对激素与相关情绪变化相关关系的研究，将对揭示激素与情绪变化的机理提供新的科学依据。本实验以此为出发点,研究啮齿类动物鼠在不同生殖周期各项指标的变化,分析各组它们血清孕激素含量与不同脑区突触外GABAA受体δ亚基的表达的相关性。 第一部分：雌性小鼠不同生殖周期大脑皮层和海马突触外GABAA受体的表达研究 目的探究雌性小鼠不同动情周期动物行为学的变化，不同生殖周期皮层及海马突触外GABAA受体的表达的变化。 方法以6周龄有完整动情周期雌性C57BL/6J小鼠实验对象,采用阴道涂片法,每天分8:00、16:00和22:00三个时间段进行动情周期的鉴别,以精确判断小鼠动情周期,筛选出具有完整生殖周期的小鼠，确定其每个周期的持续时间。并将其分为两组,即动情期组与动情间期组;Elisa检测每组小鼠血清雌孕激素含量;高架十字迷宫实验观察每组小鼠的行为学变化;免疫组织化学法观察每组小鼠海马突触外GABAA受体δ亚基的表达;分析各组小鼠血清孕激素含量与突触外GABAA受体δ亚基的表达的相关性。 结果Elisa结果显示,动情间期组雌鼠血清孕酮含量（12.47±1.89ng/mL）高于动情期组（6.02±2.20ng/mL）,而雌激素水平动情间期（157.00±21.30pg/mL）低于动情期组（185.48±35.42pg/mL）;本研究室使用的小鼠高架十字迷宫实验显示，动情期组与动情间期组小鼠在高架十字迷宫开臂、闭臂以及中央平台的活动总的活动时间无明显差异，但是在开臂中小鼠活动的次数和时间，动情间期组高于动情期组,差异具有统计学意义(P＜0.05）;免疫组化结果显示,海马脑区GABAA受体δ亚基的阳性表达动情间期组（28.93×10~(-3)±4.92×10~(-3)）高于动情期组（18.89×10~(-3)±3.74×10~(-3)）较强,差异具有统计学意义(P＜0.05），，大脑皮层GABAA受体δ亚基阳性表达较强,但动情间期组（39.84×10~(-3)±2.97×10~(-3)）与动情期组（37.31×10~(-3)±1.13×10~(-3)）无明显差异（P0.05）。动情间期与动情期海马δ亚基阳性结果IOD值与体内孕酮浓度的变化呈显著性相关,相关系数为r=0.9924。结论1.C57BL/6J小鼠平均动情周期为5.3天,小鼠血液雌激素浓度动情期组高于动情间期组，血液孕酮浓度动情间期组高于动情期组，雌、孕酮在小鼠体内随卵巢变化呈周期性的变化。2.在小鼠生理状况下,不同生殖周期雌孕激素水平会发生波动,进而影响GABAA受体δ亚基在不同生殖周期特定脑区的表达。海马脑区突触外GABAA受体δ亚基的表达在动情期低,而动情间期高,且突触外δ亚基的表达随孕酮浓度的增高而加强,两者呈线性相关。3.大脑皮层突触外GABAA受体δ亚基的表达在动情间期与动情期无明显差异。4.行为学实验显示：动情间期小鼠在高架十字迷宫上进入开臂的时间和次数大于动情期小鼠，动情间期小鼠对环境变化的适应性增高。 第二部分：大鼠大脑海马齿状回、杏仁核突触外GABAA受体表达与生殖周期雌孕激素变化相关性研究 目的明确生殖周期中卵巢甾体激素波动是否引发特定脑区神经元突触外GABAA受体表达变化及相关性。方法阴道涂片法筛选出具有完整生殖周期的大鼠，并将其分为两组，动情期组与动情间期组；Elisa检测每组大鼠血清雌孕激素含量；免疫组化法观察每组大鼠海马齿状回、杏仁核突触外GABAA受体δ亚基的表达；分析各组大鼠血清孕激素含量与突触外GABAA受体δ亚基的表达的相关性。结果血清雌激素含量动情期组(42.51±2.42)高于动情间期组(23.17±1.45)，而孕酮含量动情期组(7.41±2.86)则低于动情间期组(10.31±1.74)，组间差异具有统计学意义(P0.05)。免疫组化结果显示，海马齿状回GABAA受体δ亚基的阳性表达动情间期组(105.47±4.73)高于动情期组(79.23±2.89)，差异具有统计学意义(P0.05),杏仁核GABAA受体δ亚基的阳性表达动情间期(93.51±3.54)组高于动情期组(58.92±1.47)，差异具有统计学意义(P0.05)；海马齿状回、杏仁核突触外δ亚基阳性结果IOD值随着动情间期与动情期孕酮浓度值的增加而增加,两变量呈线性相关,相关系数分别为R=0.9866、R1=0.9876。结论动情间期高水平的孕激素可致大脑海马齿状回、杏仁核突触外GABAA受体高表达。
[Abstract]:The female reproductive cycle has periodic changes of estrogen and progesterone, especially in the pregnancy process and the large changes in the hormone after childbirth, which make all kinds of emotional disorders and various clinical symptoms in women. Some clinical symptoms are associated with the effect of progestin on the GABAA regulation system. However, the regulation of neurosteroid and its central nervous system in the body has not been unified with this kind of depression. The present study is not enough to reveal the essence of this kind of disease. This study will provide a new scientific basis for revealing the mechanism of hormonal and emotional changes. In this experiment, the changes in the various reproductive cycle indices of rodents and the correlation between the serum progesterone content and the expression of the extracellular GABAA receptor delta subunit in different brain regions were analyzed.
Part one: the expression of GABAA receptor in the cerebral cortex and hippocampus of female mice in different reproductive cycles
Objective to explore the behavioral changes of female mice in different estrous cycles, and the expression of GABAA receptor in the cortex and hippocampus of different reproductive cycles.
Methods the estrous cycle was identified by vaginal smear method with 6 weeks old estrous cycle female C57BL/6J mice. The estrous cycle was identified by 8:00,16:00 and 22:00 every day. In order to accurately determine the estrous cycle of mice, the mice with complete reproductive cycle were screened and the duration of each cycle was determined, and it was divided into two groups. The levels of estrus and estrus were observed in the estrus group and the estrus interval group; Elisa was used to detect the serum estrogen and progesterone content in each group of mice; the elevated cross maze test was used to observe the behavioral changes in each group of mice; the expression of the GABAA receptor delta subunit in the hippocampus of the hippocampus in each group of mice was observed by immunohistochemistry, and the content of progestin and the extracellular GABAA receptor delta subunit in the blood of each group were analyzed. The correlation of expression.
Results the results of Elisa showed that the content of progesterone in the serum of estrous group (12.47 + 1.89ng/mL) was higher than that of the estrus group (6.02 + 2.20ng/mL), while the estrus level (157 + 21.30pg/mL) was lower than that of the estrus group (185.48 + 35.42pg/mL). The mice elevated cross maze test in the study room showed that the estrus group was smaller than the estrus interval group. There was no significant difference in the total activity time between the open arm of the elevated cross maze, the closed arm and the central platform, but the number and time of the activity of mice in the open arm were higher than that in the estrus group. The difference was statistically significant (P < 0.05). The immunohistochemical results showed that the positive expression of the GABAA receptor delta subunit in the hippocampus was between the estrus and the estrus. The period group (28.93 x 10~ (-3) + 4.92 x 10~ (-3)) was higher than that of the estrus group (18.89 x 10~ (-3) + 3.74 x 10~ (-3)), and the difference was statistically significant (P < 0.05), and the positive expression of the GABAA receptor delta subunit in the cerebral cortex was stronger, but there was no significant difference between the estrus interval group (39.84 x 10~ (-3) + 2.97 * 2.97). The IOD value of hippocampal delta subunit positive in estrus and estrus was significantly correlated with the changes in the concentration of progesterone in the body. The correlation coefficient was r=0.9924. conclusion that the average estrous cycle of 1.C57BL/6J mice was 5.3 days, the estrus concentration in the mice was higher than that in the estrus interval group, and the concentration of the estrone concentration in the blood was higher than that in the estrus group, the female and the pregnant women. The variation of ketone in the mice changes periodically with the changes of the ovary.2.. In the physiological state of the mice, the levels of estrogen and progesterone fluctuate in the different reproductive cycle, and then affect the expression of the GABAA receptor delta subunit in the specific brain regions of the reproductive cycle. The expression of the extracellular GABAA receptor delta subunit in the hippocampus is low in the estrous stage and high in the interval of the oestrus. The expression of the extracellular delta subunit was enhanced with the increase of progesterone concentration, and there was a linear correlation between the expression of the extracellular GABAA receptor delta subunit in the.3. cerebral cortex and no significant difference between the estrus and the estrus. The.4. behavior test showed that the time and number of time and times of entering the arm of the estrus cross maze in the elevated cross maze of the estrus mice were greater than that of the estrus mice and the estrus. The adaptability of interphase mice to environmental changes was increased.
The second part: the correlation between the expression of GABAA receptor in the amygdaloid nucleus and the reproductive cycle, estrogen and progesterone in the rat dentate gyrus.
Objective to determine whether the ovarian steroid hormone fluctuations in the reproductive cycle lead to the change and correlation of the expression of extracellular GABAA receptor in specific brain neurons. Methods rats with complete reproductive cycle were screened by vaginal smear method, and were divided into two groups, estrus group and estrus interval group, and Elisa in each group of rats serum estradiol and progesterone content; Immunohistochemical method was used to observe the expression of GABAA receptor delta subunit in the dentate gyrus and the amygdala in the amygdala. The correlation between the serum progesterone content and the expression of the GABAA receptor delta subunit in the rats was analyzed. Results the serum estrogen content in the estrous group was (42.51 + 2.42) higher than that of the estrus interval (23.17 + 1.45), while the progesterone content was estrous. The period group (7.41 + 2.86) was lower than that of the estrus interval (10.31 + 1.74), and the difference between the groups was statistically significant (P0.05). The immunohistochemical results showed that the positive expression of the GABAA receptor delta subunit in the dentate gyrus of the hippocampus was (105.47 + 4.73) higher than that of the estrous phase group (79.23 + 2.89), and the difference was statistically significant (P0.05), and the GABAA receptor delta subunit of the amygdala was in the amygdala. The positive expression of the estrus interval (93.51 + 3.54) was higher than that of the estrus group (58.92 + 1.47), the difference was statistically significant (P0.05), the hippocampal dentate gyrus and the IOD value of the extracellular delta subunit of the amygdala increased with the increase of the concentration of progesterone in estrus and estrus, and the two variables were linear correlation, and the correlation coefficients were R=0.9866, R1=0.9876. Conclusion high estrus hormone can induce high expression of extra synaptic GABAA receptor in amygdaloid nucleus of dentate gyrus.
【学位授予单位】：宁夏医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R749.4
本文编号：2145119
[Abstract]:The female reproductive cycle has periodic changes of estrogen and progesterone, especially in the pregnancy process and the large changes in the hormone after childbirth, which make all kinds of emotional disorders and various clinical symptoms in women. Some clinical symptoms are associated with the effect of progestin on the GABAA regulation system. However, the regulation of neurosteroid and its central nervous system in the body has not been unified with this kind of depression. The present study is not enough to reveal the essence of this kind of disease. This study will provide a new scientific basis for revealing the mechanism of hormonal and emotional changes. In this experiment, the changes in the various reproductive cycle indices of rodents and the correlation between the serum progesterone content and the expression of the extracellular GABAA receptor delta subunit in different brain regions were analyzed.
Part one: the expression of GABAA receptor in the cerebral cortex and hippocampus of female mice in different reproductive cycles
Objective to explore the behavioral changes of female mice in different estrous cycles, and the expression of GABAA receptor in the cortex and hippocampus of different reproductive cycles.
Methods the estrous cycle was identified by vaginal smear method with 6 weeks old estrous cycle female C57BL/6J mice. The estrous cycle was identified by 8:00,16:00 and 22:00 every day. In order to accurately determine the estrous cycle of mice, the mice with complete reproductive cycle were screened and the duration of each cycle was determined, and it was divided into two groups. The levels of estrus and estrus were observed in the estrus group and the estrus interval group; Elisa was used to detect the serum estrogen and progesterone content in each group of mice; the elevated cross maze test was used to observe the behavioral changes in each group of mice; the expression of the GABAA receptor delta subunit in the hippocampus of the hippocampus in each group of mice was observed by immunohistochemistry, and the content of progestin and the extracellular GABAA receptor delta subunit in the blood of each group were analyzed. The correlation of expression.
Results the results of Elisa showed that the content of progesterone in the serum of estrous group (12.47 + 1.89ng/mL) was higher than that of the estrus group (6.02 + 2.20ng/mL), while the estrus level (157 + 21.30pg/mL) was lower than that of the estrus group (185.48 + 35.42pg/mL). The mice elevated cross maze test in the study room showed that the estrus group was smaller than the estrus interval group. There was no significant difference in the total activity time between the open arm of the elevated cross maze, the closed arm and the central platform, but the number and time of the activity of mice in the open arm were higher than that in the estrus group. The difference was statistically significant (P < 0.05). The immunohistochemical results showed that the positive expression of the GABAA receptor delta subunit in the hippocampus was between the estrus and the estrus. The period group (28.93 x 10~ (-3) + 4.92 x 10~ (-3)) was higher than that of the estrus group (18.89 x 10~ (-3) + 3.74 x 10~ (-3)), and the difference was statistically significant (P < 0.05), and the positive expression of the GABAA receptor delta subunit in the cerebral cortex was stronger, but there was no significant difference between the estrus interval group (39.84 x 10~ (-3) + 2.97 * 2.97). The IOD value of hippocampal delta subunit positive in estrus and estrus was significantly correlated with the changes in the concentration of progesterone in the body. The correlation coefficient was r=0.9924. conclusion that the average estrous cycle of 1.C57BL/6J mice was 5.3 days, the estrus concentration in the mice was higher than that in the estrus interval group, and the concentration of the estrone concentration in the blood was higher than that in the estrus group, the female and the pregnant women. The variation of ketone in the mice changes periodically with the changes of the ovary.2.. In the physiological state of the mice, the levels of estrogen and progesterone fluctuate in the different reproductive cycle, and then affect the expression of the GABAA receptor delta subunit in the specific brain regions of the reproductive cycle. The expression of the extracellular GABAA receptor delta subunit in the hippocampus is low in the estrous stage and high in the interval of the oestrus. The expression of the extracellular delta subunit was enhanced with the increase of progesterone concentration, and there was a linear correlation between the expression of the extracellular GABAA receptor delta subunit in the.3. cerebral cortex and no significant difference between the estrus and the estrus. The.4. behavior test showed that the time and number of time and times of entering the arm of the estrus cross maze in the elevated cross maze of the estrus mice were greater than that of the estrus mice and the estrus. The adaptability of interphase mice to environmental changes was increased.
The second part: the correlation between the expression of GABAA receptor in the amygdaloid nucleus and the reproductive cycle, estrogen and progesterone in the rat dentate gyrus.
Objective to determine whether the ovarian steroid hormone fluctuations in the reproductive cycle lead to the change and correlation of the expression of extracellular GABAA receptor in specific brain neurons. Methods rats with complete reproductive cycle were screened by vaginal smear method, and were divided into two groups, estrus group and estrus interval group, and Elisa in each group of rats serum estradiol and progesterone content; Immunohistochemical method was used to observe the expression of GABAA receptor delta subunit in the dentate gyrus and the amygdala in the amygdala. The correlation between the serum progesterone content and the expression of the GABAA receptor delta subunit in the rats was analyzed. Results the serum estrogen content in the estrous group was (42.51 + 2.42) higher than that of the estrus interval (23.17 + 1.45), while the progesterone content was estrous. The period group (7.41 + 2.86) was lower than that of the estrus interval (10.31 + 1.74), and the difference between the groups was statistically significant (P0.05). The immunohistochemical results showed that the positive expression of the GABAA receptor delta subunit in the dentate gyrus of the hippocampus was (105.47 + 4.73) higher than that of the estrous phase group (79.23 + 2.89), and the difference was statistically significant (P0.05), and the GABAA receptor delta subunit of the amygdala was in the amygdala. The positive expression of the estrus interval (93.51 + 3.54) was higher than that of the estrus group (58.92 + 1.47), the difference was statistically significant (P0.05), the hippocampal dentate gyrus and the IOD value of the extracellular delta subunit of the amygdala increased with the increase of the concentration of progesterone in estrus and estrus, and the two variables were linear correlation, and the correlation coefficients were R=0.9866, R1=0.9876. Conclusion high estrus hormone can induce high expression of extra synaptic GABAA receptor in amygdaloid nucleus of dentate gyrus.
【学位授予单位】：宁夏医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R749.4
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