运动性动情周期抑制大鼠卵巢激素表达的变化及差异表达基因的研究

发布时间：2018-12-13 02:02

【摘要】：目的：初步探讨(SD)雌性大鼠在长期力竭游泳运动导致动情周期抑制后卵巢相关激素的表达变化和基因表达差异,从受体和基因水平探讨运动性月经失调(AMI)的发生机制。方法：选用3月龄、健康且有正常动情周期的雌性SD大鼠22只,随机分为造模组(n=12只)和空白对照组(n=10只)。空白对照组不做任何干预,造模组每天进行力竭游泳训练,每周游泳6天,休息1天,每天上午8时对两组大鼠进行阴道涂片。当确定造模成功后处死大鼠,取卵巢进行一般形态学观察、测量并制成组织切片,利用HE染色和免疫组织化学等方法观察卵巢一般组织结构、卵巢E2、ER、PR以及血清生殖激素等的表达变化,并采用RIA技术检测血清FSH、LH、E2、P浓度；在前期试验中通过RFDD-PCR技术建立两组差异基因表达谱,并在其中选取与蛋白质的降解密切相关的泛素蛋白酶系统的相关基因,包括Psmb5、Psmd1、Psnd8、Ube3a、Usp10、Usp9x等进行定量PCR验证。结果：1.在训练过程中,造模组所有大鼠逐渐出现动情周期紊乱,同时,空白对照组大鼠动情周期保持正常。2、HE染色结果显示：在造模组,典型生长卵泡和新鲜黄体明显减少,原始卵泡、闭锁卵泡相对增加；而在空白对照组卵巢皮质中,可见大量不同阶段的卵泡,典型生长卵泡和新鲜黄体。3、卵巢E2、ER、PR免疫组化分析结果显示：造模组颗粒细胞与黄体细胞E2表达的IOD比空白组显著降低(P0.05)；与空白组相比,造模组卵巢黄体细胞中和颗粒细胞中ER表达的IOD显著降低(P0.05)；与空白组相比,造模组颗粒细胞和黄体细胞中PR表达的IOD也显著降低(P0.05)。4、血清生殖激素结果显示：与空白组相比,造模组大鼠血清E2、P、FSH、LH水平显著降低(P0.05),具有显著性差异；5、经过定量PCR验证,Psmb5、Psmd1、 Psmd8、Ube3a、Usp10、Usp9x在模型组中过表达,与RFDD-PCR测得的结果一致。结论：1、长期力竭运动可引起雌性大鼠动情周期抑制,可为游泳运动项目女运动员运动性月经失调的研究提供动物模型。2、长期力竭运动使大鼠卵巢形态学发生改变,进而抑制卵巢功能,使卵巢激素的表达发生改变,血清性激素水平下降。3、泛素蛋白酶系统中Psmb5、Psmd1、Psmd8、Ube3a、Usp10、Usp9x过表达,可能在长期力竭运动引起的雌性大鼠动情周期抑制中发挥重要的作用。
[Abstract]:Aim: to investigate the changes of ovarian related hormones and gene expression in (SD) female rats after prolonged exhaustive swimming induced estrous cycle inhibition. To explore the mechanism of (AMI) in exercise induced menstrual disorders at the receptor and gene levels. Methods: 22 healthy female SD rats were randomly divided into model group (n = 12) and blank control group (n = 10). The blank control group did not do any intervention, the model group was given exhaustive swimming training every day, swimming 6 days a week, resting for one day, and taking vaginal smear on the rats of the two groups at 8: 00 am every day. The rats were killed after the successful establishment of the model, the ovary was taken for general morphological observation, and the tissue sections were measured and made. The general structure of the ovary was observed by HE staining and immunohistochemistry, and the ovary E2ER-ER2 was observed. The expression of PR and serum reproductive hormone were detected by RIA technique. Two sets of differential gene expression profiles were established by RFDD-PCR technique in previous trials, and the genes related to the ubiquitin protease system, including Psmb5,Psmd1,Psnd8,Ube3a,Usp10, which were closely related to protein degradation, were selected. Usp9x et al were tested by quantitative PCR. Results: 1. During the course of training, all the rats in the model group showed estrous cycle disorder gradually, while the estrus cycle of the blank control group kept normal. 2 the results of HE staining showed that the typical growth follicles and fresh corpus luteum decreased obviously in the model group. Primordial follicle, atresia follicle relatively increased; In the control group, there were a large number of follicles at different stages, typical growth follicles and fresh corpus luteum. The results of PR immunohistochemical analysis showed that the expression of E2 in granulosa cells and luteal cells in model group was significantly lower than that in blank group (P0.05). Compared with the blank group, the IOD expression of ER in luteal cells and granulosa cells in the model group was significantly lower than that in the control group (P0.05). Compared with the blank group, the IOD expression of PR in granulosa cells and luteal cells in the model group was significantly lower than that in the blank group (P0.05). The level of LH was significantly decreased (P0.05). 5. The results of quantitative PCR showed that Psmb5,Psmd1, Psmd8,Ube3a,Usp10,Usp9x was overexpressed in the model group, which was consistent with the results obtained by RFDD-PCR. Conclusion: 1. Long-term exhaustive exercise can induce estrous cycle inhibition in female rats, and can provide an animal model for the study of exercise menstrual disorders of female athletes in swimming events. 2. Long-term exhaustive exercise can cause morphological changes in the ovary of rats. Furthermore, the ovarian function was inhibited, the expression of ovarian hormone was changed, the serum sex hormone level was decreased. 3. The overexpression of Psmb5,Psmd1,Psmd8,Ube3a,Usp10,Usp9x in the ubiquitin protease system. It may play an important role in the inhibition of estrous cycle induced by long-term exhaustive exercise in female rats.
【学位授予单位】：成都体育学院
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R87

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