雌激素对L型钙通道调控的研究

发布时间：2018-05-25 15:38

本文选题：神经元培养 + 雌激素　；参考：《重庆医科大学》2014年硕士论文

【摘要】：目的：明确雌激素对L型钙通道电流的调控作用以及对L型钙通道蛋白水平的影响。方法： 1.将新生Sprague-Dawley（SD）大鼠皮层神经元培养，定期倒置显微镜观察细胞形态及走形，第7天行免疫荧光神经元鉴定。 2.以新生大鼠原代皮层神经元为研究对象，培养至7天左右时给予干预，实验设溶剂对照组及雌激素E2（1umol/L）组，干预72小时后采用全细胞膜片钳技术测定L型钙通道电流。 3.培养至第7天左右神经元给予干预，，实验设溶剂对照组、雌激素受体拮抗剂ICI182780（简称ICI，100nmol/L）组、雌激素E2（1umol/L）组及雌激素E2（1umol/L）加雌激素拮抗剂ICI182780（100nmol/L）组，干预72小时后提取细胞蛋白用蛋白免疫印记Western blot方法检测神经元总Cav1.2量的变化。结果： 1、培养第7天的细胞胞体饱满，折光性强，走形良好，轴突密集形成网状结构，免疫荧光结果显示大部分细胞MAP-2染色阳性（红染），为神经元细胞，形态良好。 2.全细胞膜片钳结果显示雌激素处理组L型钙通道电流较对照组神经元电流明显减少（P＜0.05）， 3.蛋白免疫印迹Westernblot结果提示雌激素处理后Cav1.2的量明显减少（P＜0.01），正常组与雌激素受体拮抗剂ICI、雌激素加ICI组无明显差异（P＞0.05）。结论： 1.原代皮层神经元细胞培养成功。 2.雌激素处理可以减少神经元L型钙通道电流。 3.雌激素处理减少了原代皮层神经元LTCC蛋白Cav1.2
[Abstract]:Objective: To investigate the effect of estrogen on L type calcium channel current and on L type calcium channel protein level. Methods: 1. The cortical neurons of newborn Sprague-Dawley (SD) rats were cultured, and the morphology and aberration of the neurons were observed by inverted microscope. The neurons were identified by immunofluorescence on the 7th day. 2. Primary cortical neurons of newborn rats were taken as the study object. After 7 days of culture, the primary cortical neurons were treated with solvent control group and estradiol / L group. After 72 hours of intervention, L-type calcium channel currents were measured by whole-cell patch clamp technique. 3. The neurons were cultured for 7 days. The rats were divided into solvent control group, estrogen receptor antagonist ICI182780 (ICI182780) group, estrogen E2O1umol/ L group and estrogen E2O1umolr-L + ICI182780nmol / L group. After 72 hours of intervention, the changes of the total Cav1.2 of neurons were detected by protein immunoimprinted Western blot. Results: 1. On the 7th day of culture, the cells had full body, strong refraction, good shape, dense axons formed reticular structure, and the immunofluorescence results showed that most of the cells were positive for MAP-2 staining (red staining, neuronal cells, good morphology). 2. The results of whole-cell patch clamp showed that the L-type calcium channel current in estrogen treated group was significantly lower than that in control group (P < 0.05). 3. Western blot Westernblot results showed that the amount of Cav1.2 decreased significantly after estrogen treatment (P < 0.01). There was no significant difference between normal group and estrogen receptor antagonist ICI, estrogen plus ICI group (P > 0.05). Conclusion: 1. Primary cortical neurons were successfully cultured. 2. Estrogen treatment reduced L-type calcium channel currents in neurons. 3. Estrogen treatment reduced LTCC Cav1.2 in primary cortical neurons
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R741

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