氯米芬对雌性SD大鼠子宫内膜基因组表达影响及机制研究

发布时间：2018-05-10 17:49

本文选题：枸橼酸氯米芬 + 子宫内膜　；参考：《昆明理工大学》2014年硕士论文

【摘要】：研究目的：枸橼酸氯米芬(Clomiphene Citrate, CC)由于具有价格低廉,使用方便,促排卵效果好等优点,目前仍是微刺激方案中的一线药物,研究表明,CC可能会抑制子宫内膜的增殖,降低子宫内膜的厚度,对子宫内膜容受性可能有不良影响,从而使试管婴儿临床妊娠率偏低。但CC影响子宫内内膜容受性的分子机制至今尚不清楚。为了弄清CC如何通过抑制子宫内膜细胞的增殖影响子宫内膜容受性,以提高低刺激方案的临床成功率。本研究采用CC对雌性SD大鼠给药,然后对CC治疗后雌性大鼠子宫内膜基因组表达的变化进行研究,探讨CC影响子宫内膜增殖的相关机制。实验方法：本研究以200-250g体重的雌性SD大鼠为研究对象,对大鼠进行枸橼酸氯米芬灌胃给药,并根据不同的给药时间将大鼠分为六组,每组都设置相应的空白对照组(用等量的生理盐水代替氯米芬)。给药结束后取其子宫组织切片进行HE染色,观察子宫内膜的厚度变化。根据其结果我们选用了给药6天组的5只大鼠以及相应的5只同期空白组大鼠,提取子宫组织总RNA,然后采用了Qiagen公司的大鼠雌激素受体信号通路PCR分析芯片进行PCR阵列分析。分析后我们发现在CC给药组大鼠子宫中c-myc出现了高表达。因此我们将一批大鼠分为5个组(分别为：CC组、CC+E2组、CC+HMG组、CC+E2+HMG组和空白组,给药剂量分别为：CC:1mg/kg体重；HMG:0.75IU/只；E2:10mg/kg体重,给药时间都为6天),取子宫组织做Western Blot检测c-myc表达情况。由于NCOA1位于c-myc信号通路上游,与雌激素受体共同调控了c-myc的表达,我们对枸橼酸氯米芬不同给药时间的SD大鼠子宫中NCOA1表达进行了Western Bloting检测。实验结果：枸橼酸氯米芬确实导致雌性SD大鼠子宫内膜厚度降低,而给药第6天,给药组子宫内膜厚度与空白组子宫内膜厚度相差最大。枸橼酸氯米芬能够上调子宫中c-myc表达,且c-myc在不同给药组中均高表达。而不同给药时间的SD大鼠子宫中NCOA1表达均出现下调,且NCOA1受大鼠发情周期影响。同时我们发现枸橼酸氯米芬使血清中E2没有显著影响但CC+E2+HMG组相比对照组出现下调,而枸橼酸氯米芬对P、FSH和LH血清基本无显著影响。结论：1.枸橼酸氯米芬能够降低子宫内膜厚度,影响子宫内膜容受性；2.枸橼酸氯米芬上调子宫内c-myc mRNA和蛋白水平,同时下调了NCOA1蛋白水平;3.枸橼酸氯米芬使血清中E2没有显著影响但CC+E2+HMG组相比对照组出现下调,而枸橼酸氯米芬对P、FSH和LH血清无显著影响。
[Abstract]:Objective: clomiphene citrate (CCC) is still a first-line drug in microstimulation because of its advantages of low cost, convenient use and good effect of ovulation promotion. Studies have shown that CCCC may inhibit the proliferation of endometrium. Lowering the thickness of endometrium may have a negative effect on endometrial receptivity, which makes the clinical pregnancy rate of IVF low. However, the molecular mechanism of CC affecting endometrial receptivity is still unclear. In order to understand how CC affects endometrial receptivity by inhibiting endometrial cell proliferation in order to improve the clinical success rate of low stimulation regimen. In this study, CC was administered to female SD rats, and then the changes of endometrial genomic expression in female rats after CC treatment were studied to explore the mechanism of CC affecting endometrial proliferation. Methods: in this study, the female SD rats with 200-250 g body weight were given clomiphene citrate intragastric administration, and were divided into six groups according to different administration time. Each group was given a corresponding blank control group (the same amount of normal saline instead of clomiphene. The thickness of endometrium was observed by HE staining. According to the results, we selected 5 rats in the 6-day group and 5 rats in the corresponding blank group to extract total RNAs from uterine tissue, and then analyzed the PCR array by using the PCR analysis chip of estrogen receptor signaling pathway of Qiagen company in rats. After analysis, we found that there was a high expression of c-myc in the uterus of rats treated with CC. Therefore, we divided a group of rats into five groups (1: CC group, CC E2 group, CC HMG group, CC E 2 HMG group and blank group, respectively). The dosage of the drug was 1 mg / kg / 1 mg / kg, respectively. The administration time was 6 days. The expression of c-myc in uterine tissues was detected by Western Blot. Because NCOA1 is located upstream of c-myc signaling pathway and regulates the expression of c-myc together with estrogen receptor, we detected the expression of NCOA1 in the uterus of SD rats with different administration time of clomiphene citrate by Western Bloting. The results showed that clomiphene citrate did decrease the endometrial thickness of female SD rats, but on the 6th day, the difference between the endometrial thickness in the administration group and that in the blank group was the greatest. Clomiphene citrate could upregulate the expression of c-myc in uterus, and c-myc was overexpressed in different groups. However, the expression of NCOA1 was down-regulated in the uterus of SD rats with different administration time, and NCOA1 was affected by the estrus cycle of rats. At the same time, we found that clomiphene citrate had no significant effect on serum E2, but decreased in CC E2 HMG group compared with control group, but clomiphene citrate had no significant effect on serum level of HMG and LH. Conclusion 1. Clomiphene citrate can reduce endometrial thickness and affect endometrial receptivity. Clomiphene citrate upregulated the levels of c-myc mRNA and protein in uterus and down-regulated the level of NCOA1 protein. Clomiphene citrate had no significant effect on E2 in serum, but decreased in CC E2 HMG group compared with control group, but clomiphene citrate had no significant effect on FSH and LH serum.
【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R714.8

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