新生期不同剂量双酚A暴露对雌性大鼠青春发育的影响

发布时间：2018-06-03 10:01

本文选题：BPA + 新生雌性SD大鼠　；参考：《苏州大学》2014年硕士论文

【摘要】：目的：研究新生期不同剂量双酚A（bisphenol A，BPA）暴露对雌性SD大鼠阴道开口时间（vaginal opening day，VOD）、下丘脑kiss-1mRNA及其蛋白kisspeptin表达和子宫、卵巢雌激素受体α（Estrogen Receptor，ERα）mRNA、雌激素受体β（Estrogen Receptor，ERβ）mRNA及其蛋白表达的影响。方法：将新生雌性SD大鼠随机分为6组，即空白对照组、溶剂组、17β-雌二醇组（17β-estradiol，E2，10μg d-1）、低剂量BPA组（25μg kg-1d-1）、中剂量BPA组（50μg kg-1d-1）和高剂量BPA组（250μg kg-1d-1），在生后第0-6天（postnatal day0-6，PND0-6）经皮下注射每日给药。记录VOD，并于当天处死大鼠，取出下丘脑、子宫、卵巢并称重，计算下丘脑、子宫、卵巢脏器系数。对下丘脑组织行免疫组化检测观察kisspeptin定位，子宫、卵巢行HE染色观察形态变化。通过Real—Time PCR的方法测定下丘脑kiss-1mRNA和子宫、卵巢中ERα、ERβ mRNA表达量。通过Western-blot的方法测定下丘脑中kisspeptin及子宫、卵巢中ERα、ERβ蛋白表达量。结果：1.对体重的影响：自PND0至PND6每日对大鼠进行皮下注射方式的药物干预，对照组与各干预组间大鼠PND0与PND6体重未有明显差异（P0.05）。2. VOD记录显示与对照组相比，E2组、50μg kg-1d-1和250μg kg-1d-1BPA组VOD较对照组提前（P0.05）；溶剂组、25μg kg-1d-1BPA组VOD与对照组相比未有显著差异（P0.05）。3.下丘脑免疫组化结果提示kisspeptin多位于神经细胞上。4. HE染色结果显示空白对照组、溶剂组、25μg kg-1d-1、50μg kg-1d-1和250μg kg-1d-1BPA组子宫肌层厚度、内膜厚度较E2组厚，卵巢卵泡发育度较E2组高。5.脏器系数结果显示，各干预组下丘脑脏器系数与对照组相比无显著差异（P0.05），但E2组子宫、卵巢脏器系数较对照组明显降低（P0.05），溶剂组、BPA干预组较对照组无明显改变（P0.05）。6. RT-PCR结果显示，，溶剂组、25μg kg-1d-1、、50μg kg-1d-1和250μg kg-1d-1BPA组下丘脑kiss-1mRNA及子宫、卵巢ER mRNA表达水平较对照组未有明显差异（P0.05）； E2组下丘脑kiss-1、子宫ERα、卵巢ERβ mRNA表达水平较对照组明显降低（P0.05），子宫ERβ、卵巢ERα mRNA水平较对照组未有明显差异（P0.05）。7. Western-Blot结果显示，溶剂组、25μg kg-1d-1、50μg kg-1d-1和250μg kg-1d-1BPA组下丘脑kisspeptin及子宫、卵巢ER蛋白水平较对照组未有明显差异（P0.05）； E2组下丘脑kisspeptin、子宫ERα、卵巢ERβ蛋白水平较对照组明显降低（P0.05），子宫ERβ、卵巢ERα蛋白水平较对照组未有明显差异（P0.05）。结论：①每日皮下注射操作未对注射药物期间大鼠基本体重增长产生明显影响。②下丘脑神经细胞上有kisspeptin表达。③新生期50μg kg-1d-1、250μg kg-1d-1BPA暴露可引起大鼠青春期启动提前。④上述青春期启动提前并没有通过kiss-1基因表达改变来实现，且未对其青春期启动时下丘脑、子宫、卵巢脏器系数，子宫、卵巢发育形态及ER表达产生明显影响。⑤新生期溶剂及25μg kg-1d-1BPA暴露未引起大鼠青春期启动提前。⑥新生期10μg d-1E2暴露可引起大鼠青春期启动提前，且启动时子宫、卵巢发育程度降低，子宫、卵巢脏器系数、下丘脑kiss-1mRNA、子宫ERα mRNA、卵巢ERβ mRNA表达量及其蛋白均较对照组明显降低。
[Abstract]:Objective: To study the vaginal opening time (vaginal opening day, VOD) of female SD rats exposed to different doses of bisphenol A (bisphenol A, BPA), and the kisspeptin expression of kiss-1mRNA and its protein in the hypothalamus and the uterus, ovarian estrogen receptor alpha, estrogen receptor beta and its protein expression. Influence.
Methods: the newborn female SD rats were randomly divided into 6 groups, namely the blank control group, the solvent group, the 17 beta estradiol group (17 beta -estradiol, E2,10 mu g D-1), the low dose BPA group (25 mu g kg-1d-1), the medium dose BPA group (50 mu g kg-1d-1) and the high dose BPA group (250 mu), and the daily delivery was recorded by subcutaneous injection on the 0-6 day after birth. OD, the rats were killed on the same day, and the hypothalamus, uterus, ovary and weight were taken out. The hypothalamus, uterus, and ovarian organ coefficient were calculated. The hypothalamus was detected by immunohistochemical staining, and the morphology of the uterus and ovary were observed by HE staining. The kiss-1mRNA and uterus in the hypothalamus, ER a, ER beta m in the ovary were measured by Real Time PCR. The expression level of RNA was measured by Western-blot. The expression level of ER and ER beta in kisspeptin and uterus and ovary of hypothalamus were measured.
Results: the effect of 1. on weight: the intervention of subcutaneous injection of subcutaneous injection from PND0 to PND6, and there was no significant difference in the weight of PND0 and PND6 between the control group and the intervention group (P0.05).2. VOD record. Compared with the control group, the group E2, the 50 micron kg-1d-1 and the 250 mu g kg-1d-1BPA group were earlier than the control group; the solvent group, 25 There was no significant difference in VOD between the G kg-1d-1BPA group and the control group (P0.05) the results of the immunohistochemical staining of the hypothalamus in.3. showed that kisspeptin was mostly located on the nerve cells and the.4. HE staining results showed the blank control group, the solvent group, the 25 micron kg-1d-1,50 mu g kg-1d-1 and the 250 Mu uterine myometrium thickness, the thickness of the endometrium was thicker than that of the ovarian follicle development. The higher.5. organ coefficient compared with the E2 group showed that the hypothalamic organ coefficient of the intervention group had no significant difference compared with the control group (P0.05), but the uterus, the ovarian organ coefficient of the E2 group was significantly lower than the control group (P0.05), the solvent group and the BPA intervention group had no significant changes (P0.05).6. RT-PCR results (P0.05), the solvent group, 25 mu g kg-1d-1, 50 micron g, and 2. The expression level of ER mRNA in the hypothalamus of the 50 g kg-1d-1BPA group was not significantly different than that of the control group (P0.05). The level of KiSS-1 in the hypothalamus, ER alpha and ER beta in the E2 group was significantly lower than that of the control group (P0.05), and there was no significant difference between the uterus and the ovary of the control group. The level of kisspeptin and uterus of the hypothalamus in the group of solvent, 25 g kg-1d-1,50 g kg-1d-1 and 250 mu g kg-1d-1BPA group was not significantly different from that of the control group (P0.05), and the kisspeptin of the hypothalamus in the E2 group, the uterus ER alpha and the ovarian beta protein level were significantly lower than those in the control group. Significant difference (P0.05).
Conclusion: (1) daily subcutaneous injection did not significantly affect the basic weight growth of rats during the period of injection. (2) there was kisspeptin expression on the hypothalamus nerve cells. (3) 50 g kg-1d-1250 mu g kg-1d-1BPA exposure in the newborn period could lead to early puberty initiation in rats. (4) the early puberty start up did not pass through the KiSS-1 gene expression. Change to achieve, and did not have a significant effect on the hypothalamus, uterus, ovarian organ coefficient, uterus, ovarian development morphology and ER expression during puberty. 5. The neonatal period solvent and 25 g kg-1d-1BPA exposure did not lead to early puberty initiation. The degree of uterine and ovarian development decreased, the uterus, the ovarian organ coefficient, the kiss-1mRNA of the hypothalamus, the ER alpha mRNA of the uterus, the expression of ER beta mRNA in the ovary and their proteins were significantly lower than those of the control group.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R714.2

【参考文献】