双酚A对大鼠卵巢甾体激素生成影响的初步研究

发布时间：2018-07-04 22:18

本文选题：双酚A + 卵泡内膜细胞　；参考：《南京医科大学》2007年硕士论文

【摘要】： 目的：双酚A(Bisphenol A，BPA)是一种常见的环境内分泌干扰物(Environmental endocrine disruptors，EEDs)，对女性生殖内分泌系统有潜在的影响。本文通过大鼠卵巢内膜间质细胞及颗粒细胞的原代培养体系，研究BPA对卵泡内膜细胞睾酮生成和对细胞色素P45017α羟化酶(P450c17)、胆固醇侧链裂解酶(P450scc)和类固醇激素急性调节蛋白(StAR)mRNA的影响；以及研究BPA对颗粒细胞激素生成及芳香化酶(P450arom)、胆固醇侧链裂解酶(P450scc)和类固醇激素急性调节蛋白(StAR)mRNA的影响，探讨BPA对女性生殖系统激素生成的影响机制，论证环境内分泌干扰物BPA影响女性生殖系统生长，发育，疾病产生的可能机理。方法：分别采用大鼠的卵泡内膜间质细胞、颗粒细胞进行体外原代培养，培养72 h后以0、10~(-7)、10~(-6)、10~(-5)、10~(-4) M五个不同浓度的BPA作用细胞，继续培养48 h，收集细胞培养液，用超敏感的固相放射免疫方法测定内膜间质细胞生成的睾酮浓度及颗粒细胞分泌的雌二醇、孕酮的水平。沉淀细胞，提取细胞总RNA，，反转录为cDNA，采用荧光实时RT-PCR(TaqMan.assay)方法测定卵泡内膜细胞P450c17a、P450Scc二种酶和StAR mRNA表达水平及颗粒细胞P450arom、P450Scc二种酶和StAR mRNA表达水平的变化。结果： BPA刺激原代培养的卵泡内膜间质细胞睾酮的分泌，培养液中睾酮水平显著升高(p＜0.05)。睾酮合成相关的酶的基因表达水平也升高，在BPA 10~(-4)M剂量组出现统计学意义(p＜0.05)。StAR的表达也显著增加(p＜0.05)。原代培养的颗粒细胞孕酮分泌量随BPA浓度的增加而增加(p＜0.05)，但在10~(-4) M剂量组其浓度出现陡降(p＜0.01)；而雌二醇的分泌量则随BPA浓度的增加而下降(p＜0.01)。雌孕激素生成相关基因P450arom mRNA表达显著降低；P450Scc表达量在10~(-7) M到10~(-5)M组逐渐升高(p＜0.05)，而10~(-4)组却出现显著降低(p＜0.01)。StAR mRNA表达则有不同程度的增高(p＜0.05)。结论： BPA能直接作用于卵巢内膜间质细胞及颗粒细胞：通过诱导P450c17a，P450Scc等相关酶的表达增加、以及增加类固醇合成的限速因子-StAR的表达，增加内膜细胞睾酮的合成；通过作用于P450arom，P450Scc等相关酶的表达以及类固醇合成的限速因子-StAR的表达而干扰颗粒细胞雌孕激素的分泌。故生物体如果暴露于一定量的BPA，很可能导致生殖内分泌系统的紊乱从而产生一些内分泌疾病。其中一部分原因可能是通过影响卵巢激素生成酶的表达而造成女性卵巢激素生成水平的异常，从而使下丘脑-垂体-卵巢轴的正常调节机制发生改变所造成的。
[Abstract]:Objective: bisphenol A (BPA) is a common environmental endocrine disruptor (endocrine), which has a potential effect on female reproductive endocrine system. The primary culture system of rat endometrial stromal cells and granulosa cells was used. To study the effects of BPA on testosterone production and mRNA expression of cytochrome P45017 伪 -hydroxylase (P450c17), cholesterol side chain lyase (P450 SCC) and steroid hormone acute regulatory protein (star) in endometrial follicle cells. The effects of BPA on granulosa hormone production and aromatase (P450 aromatase), cholesterol side chain lyase (P450 SCC) and steroid hormone acute regulatory protein (star) mRNA were studied. The possible mechanism of environmental endocrine disruptor BPA affecting the growth, development and disease of female reproductive system was demonstrated. Methods: rat endometrial stromal cells and granulosa cells were cultured in vitro. After 72 hours of culture, the cells were treated with 10 ~ (-7) 10 ~ (-6) 10 ~ (-5) 10 ~ (-4) M BPA. After 48 hours of culture, the cell culture medium was collected, and the concentration of testosterone produced by endometrial stromal cells and the levels of estradiol and progesterone secreted by granulosa cells were measured by hypersensitive solid phase radioimmunoassay. The total RNAs were extracted from the cells and reversely transcribed into cDNA.The expression levels of two enzymes and star mRNA and the expression levels of P450aromonas P450Scc and star mRNA were measured by TaqMan.Assay in the endometrial cells of follicle. The expression of P450c17aSc and star mRNA in granulosa cells were measured by TaqMan.Assay. The expression of P450c17aFSC and P450Scc mRNA in granulosa cells were determined by TaqMan.Assay. Results: BPA stimulated the secretion of testosterone in primary cultured endometrial stromal cells, and the level of testosterone in culture medium was significantly increased (p < 0.05). The gene expression level of testosterone synthesis-related enzymes was also increased, and the expression of star was significantly increased in BPA 10 ~ (-4) M group (p < 0.05) .Star expression was also significantly increased in BPA 10 ~ (-4) M group (p < 0.05). Progesterone secretion of primary cultured granulosa cells increased with the increase of BPA concentration (p < 0.05), but decreased sharply in 104M group (p < 0.01), while that of estradiol decreased with the increase of BPA concentration (p < 0.01). The expression of P450arom mRNA decreased significantly in the 10 ~ (-7) M to 10 ~ (-5) M groups (p < 0.05), but decreased significantly in the 10 ~ (-4) group (p < 0.01). The star mRNA expression increased in varying degrees (p < 0.05). Conclusion: BPA can directly act on stromal cells and granulosa cells of ovary. By inducing the expression of P450 SCC and other related enzymes, and increasing the expression of steroid synthesis-limiting factor -Star, BPA can directly affect the expression of P450 SCC and P450 SCC in endometrial stromal cells and granulosa cells. Increase the synthesis of testosterone in endometrial cells and interfere with the secretion of estrogen and progesterone in granulosa cells by acting on the expression of P450 SC and steroid biosynthesis limiting factor -Star. Therefore, if the organism is exposed to a certain amount of BPA, it may lead to the disorder of reproductive endocrine system and some endocrine diseases. Some of the reasons may be caused by the abnormal level of female ovarian hormone production by affecting the expression of ovarian hormone producing enzyme, which may result in the change of the normal regulation mechanism of hypothalamus-pituitary-ovary axis.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R363

【相似文献】