双酚A对新生小鼠始基卵泡体外生长的影响及作用机制研究

发布时间：2018-03-21 01:01

  本文选题：双酚A　切入点：卵巢培养　出处：《第二军医大学》2014年硕士论文　论文类型：学位论文

【摘要】：[研究背景及目的] 内分泌干扰物(Endocrine disruptor chemicals, EDCs)是一种外源性的化合物,能影响人类和动物体内的内分泌系统,进而影响相应器官的功能,甚至是下一代的健康。因其在自然界的广泛存在,以及对健康的影响,近几年,EDCs受到广泛的关注。 双酚A(Bisphenol A, BPA)是目前使用量最大的内分泌干扰物,在许多常见消费类产品中存在,包括塑料(为一种聚合物,即聚碳酸酯塑料)、聚氯乙烯(PVC)、食品包装、牙科密封剂等。人类可以通过饮食、空气吸入以及皮肤接触吸收BPA。90%的抽样人群尿液中可检测出双酚A,50%的人群血液中可检测到,美国、德国、中国以及一些其它亚洲国家都得到相似的结果。更引起人们注意的是,即使很小剂量的BPA都可引起人类的不良反应,因此,多个国家均颁布法律条例来严格控制BPA的用量。然而,BPA对人类健康的影响已然存在,只有明确其所致疾病以及致病机制,才能更好的预防和治疗环境相关疾病。 大量研究显示,双酚A可导致多囊卵巢综合征(Polycystic Ovary Syndrome, PCOS)、不孕症、流产、代谢综合征等疾病。双酚A对人类的健康影响是复杂的、全身的、涉及多个器官,特别是生殖系统。无论是女性还是男性,其生殖系统都容易受到双酚A的影响。双酚A能降低男性精子的质量、引起精原细胞的凋亡；而对于女性卵巢而言,双酚A不仅影响发育卵泡的成熟与排出,还干扰颗粒细胞、卵泡膜细胞中性激素合成、分泌及代谢相关的酶活性,然而,双酚A对始基卵泡的研究还很少。 始基卵泡是卵泡发育的起始阶段,也是女性生育功能的储备池。既往对始基卵泡的相关研究很少,近几年,人们逐渐认识了始基卵泡发育与闭锁的过程,其中PTEN/PI3K途径对于始基卵泡池的维持其重要的调节作用。出生后,人类的始基卵泡不再增加,而是逐渐减少。始基卵泡的减少速度是恒定的,无论是减少过快或者减少过慢,都有可能影响后续卵泡的发育以及整体的卵巢功能。许多因素可影响始基卵泡的数量,特别是环境化合物,如VCD(4-vinylcyclohexene diepoxide)、二甲基苯芘蒽(7,12-dimethylbenz [a] anthracene,DMBA)等。已有研究显示,新生鼠注射双酚A能显著减少始基卵泡的数量而增加初级卵泡的数量,在新生羊中也观察到相同结果。然而,卵巢中卵泡生长的异质性明显,且卵泡细胞内的分子改变受卵巢取出时间的影响较大。因此,本课题采用的是新生小鼠整体卵巢体外培养体系,可以控制外界环境对卵泡的生长,使得卵巢发育同步化,减少个体差异,可以作为研究卵泡生长与凋亡的良好载体。本研究将利用体外卵巢培养体系,进一步验证双酚A对始基卵泡的影响,同时探索双酚A作用于始基卵泡的机制。 [研究方法] 1.体外新生小鼠卵巢组织培养体系的建立及其对始基卵泡生长的影响 取新生4天C57BL/6小鼠整体卵巢组织,进行体外培养,并与体内同等时间卵巢生长相比较。光镜下计量卵巢的平均直径；通过HE染色观察培养0天、5天、10天卵巢组织的病理学改变并计数各级卵泡；Ki-67及caspase3免疫组化分析体外培养卵巢卵泡的凋亡与生长情况。 2.观察双酚A对始基卵泡生长的影响 将卵巢组织随机分为4组,分别为：1、对照组(DMSO);2、低浓度双酚A组(0.1μM)；3、中等浓度双酚A组(1μM)；4、高浓度双酚A组(10μM)。通过HE染色比较各组卵巢的病理改变,并计数各级卵泡；通过Ki-67及caspase3免疫组化分析各组卵泡的生长与凋亡状况。 3.探索双酚A对卵巢组织PI3K/Akt途径的影响 检测4组卵巢组织中PI3K/Akt途径上关键物质-PI3K、Akt的表达；用Realtime-PCR检测mRNA水平上的表达；用Western-blot检测定量蛋白水平的表达。以PI3K抑制剂阻断PI3K/Akt途径后,HE染色计数初级卵泡数量。 4.统计学处理 各数据均使用SPSS20.0进行统计分析,并用GraphPad Prism5.0软件制作统计图表。每组数值以平均值士标准差表示,计数资料及方差不齐的资料采用非参数秩和检验(Kruskal-Wallis)进行分析总体差异,随后将秩和检验形成的新的秩变量作单因素方差分析及Dunnett法进行组间比较。其余计量资料均采用one-way ANOVA分析及Dunnett法或Turkey HSD法进行组间比较。P0.05认为差异有统计学意义。 [研究结果] 1.体外无血清培养条件下的始基卵泡生长与体内生长相似 体外培养的卵巢平均直径略小于体内生长卵巢,但差异无统计学意义；HE染色见体外培养卵巢组织病理学无明显改变,于培养第10天始基卵泡减少明显,具统计学意义,初级卵泡与次级卵泡数量均无明显差异。Ki-67及caspase3免疫组化提示体外培养卵巢凋亡增加,有统计学意义,主要为始基卵泡凋亡；卵泡生长无明显差异。 2.双酚A通过过度激活始基卵泡发育减少始基卵泡池数量 各浓度组双酚A与对照组相比,HE染色未见明显病理学改变,1μM、10μM双酚A均明显下降,初级卵泡数量增加,两者差异均有统计学意义。而0.1μ M双酚A组始基卵泡数量减少与初级卵泡数量增加差异无统计学意义。各组卵巢次级卵泡数与卵泡总数差异无统计学意义。Ki-67及caspase3免疫组化提示各浓度组双酚A均显著增加卵泡Ki-67表达,减少caspase3表达,提示卵泡生长增加而凋亡减少。 3.双酚A通过激活PI3K/Akt途径促进始基卵泡发育 各浓度双酚A组显著增加PI3K的mRNA和蛋白表达,以及磷酸化Akt的蛋白表达。应用PI3K抑制剂阻断PI3K/Akt途径,对照组与双酚A组的初级卵泡数量均明显下降,且含有PI3K抑制剂的对照组与双酚A组两者差异无统计学意义。 [研究结论] 体外小鼠卵巢整体组织培养体系中始基卵泡发育速度与小鼠体内始基卵泡发育相似,是研究影响始基卵泡闭锁与发育的良好载体；双酚A可显著减少体外培养的始基卵泡数量,增加初级卵泡的数量。而这种作用是双酚A过度激活始基卵泡发育、减少始基卵泡凋亡的共同结果；双酚A主要通过促进PI3K/Akt途径表达而发挥促进始基卵泡发育的作用。然而,双酚A如何作用于PI3K/Akt途径,需要进一步探索研究。
[Abstract]:[research background and purpose]
Endocrine disruptors (Endocrine disruptor chemicals, EDCs) is a kind of exogenous compounds can affect the endocrine system of human and animal body, and the corresponding impact of organ function, and even the next generation of health. Because of its widely exist in nature, as well as the impact on health, in recent years, EDCs has been widely pay attention to.
Bisphenol A (Bisphenol A BPA) is currently the largest use of endocrine disruptors, present in many common consumer products, including plastic (as a polymer, i.e. polycarbonate plastic), polyvinyl chloride (PVC), food packaging, dental sealants. Humans can through diet, air intake and urine sample of people in contact with skin the absorption of BPA.90% can be detected in bisphenol A, can be detected, 50% of the population in the blood of United States, Germany, China and some other Asian countries have obtained similar results. More attention is that even small doses of BPA can cause adverse reactions, so human, many countries have enacted laws regulations to strictly control the amount of BPA. However, the effect of BPA on human health is already exist, if only the disease caused by pathogenic mechanism and related diseases, prevention and treatment of the environment can be better.
Many studies show that bisphenol A can lead to polycystic ovary syndrome (Polycystic Ovary, Syndrome, PCOS), infertility, abortion, metabolic syndrome and other diseases. The effects of bisphenol A on human health is complex, systemic, involving multiple organs, especially reproductive system. Either female or male reproductive system vulnerable to the effects of bisphenol A. The quality of bisphenol A can reduce sperm, induce apoptosis of spermatogonia; and for the female ovary, not only affect the development of bisphenol A in the mature follicle and discharge, also interfere with granulosa cells, theca cells and the secretion of sex hormone synthesis, however, metabolism related enzyme activity, and bisphenol A study of the primordial follicles are rare.
Is the initial stage of primordial follicle development, and reproductive function. Previous research on the pool of primordial follicles rarely, in recent years, people gradually realized the process of primordial follicle development and atresia, the PTEN/PI3K pathway for the maintenance of the primordial follicle pool its important role. After birth. Human primordial follicle is not increased, but decreased gradually. The decrease speed is constant primordial, either too fast or too slow to reduce reduction, are likely to affect subsequent follicular development and overall ovarian function. Many factors can affect the number of primordial follicles, especially environmental compounds, such as VCD (4-vinylcyclohexene diepoxide), two methyl benzene pyrene (7,12-dimethylbenz [a] anthracene, DMBA). Studies have shown that injection of bisphenol A in neonatal rats can significantly reduce the number of primordial follicles and primary follicles increased the number of Amount in newborn sheep was also observed in the same results. However, the heterogeneity of follicle growth significantly in ovarian follicular cells, and within the molecular changes affected by ovary out of time. Therefore, this thesis adopts the system of mice were cultured in vitro can control the growth of the whole ovary, external environment of the follicle, the ovary the development of synchronization, reduce the individual differences, can be used as a good carrier of follicle growth and apoptosis. In this study, using in vitro culture system of ovary, further verify the effects of bisphenol A on the primordial follicle, while exploring the mechanism of bisphenol A in primordial follicles.
[research method]
Establishment of tissue culture system of 1. neonatal mouse ovary and its effect on the growth of primordial follicle
The 4 day newborn C57BL/6 mice ovarian tissue were cultured in vitro and in vivo growth compared with the same time. The average diameter of ovarian under light microscope by measuring the ovary; HE staining cultured for 0 days, 5 days, 10 days of pathological ovarian tissue pathology and counting of follicles; Ki-67 and Caspase3 immunohistochemical analysis in vitro apoptosis and growth of ovarian follicles.
2. effect of bisphenol A on the growth of primordial follicles
The ovarian tissues were randomly divided into 4 groups, respectively: 1, the control group (DMSO); 2, low concentration of bisphenol A group (0.1 M); 3, moderate concentration of bisphenol A group (1 M); 4, the high concentration of bisphenol A group (10 M). The pathological changes of ovary were compared by HE staining, and counting the number of follicles; through Ki-67 and Caspase3 immunohistochemical analysis of follicle growth and apoptosis status in the group.
3. explore the effect of bisphenol A on the PI3K/Akt pathway of ovarian tissue
The expression of -PI3K and Akt in the PI3K/Akt pathway was detected in 4 groups of ovarian tissues. The expression level of mRNA was detected by Realtime-PCR, and the expression level of quantitative protein was detected by Western-blot. After blocking PI3K/Akt pathway with PI3K inhibitor, the number of primary follicles was counted by HE staining.
4. statistical treatment
The data were analyzed by SPSS20.0, and use GraphPad Prism5.0 software to make statistics. Each value to the average + standard error of the mean and variance of count data, missing data using the nonparametric Wilcoxon test (Kruskal-Wallis) were analyzed with the overall difference, after the rank sum test a new rank as a single variable ANOVA and Dunnett were compared between the two groups. One-way ANOVA analysis and Dunnett method or Turkey HSD method are used in the rest of the measurement data were compared between the two groups of.P0.05 that the difference was statistically significant.
[results]
1. the growth of primordial follicle in vitro is similar to the growth of the body under the conditions of serum-free culture in vitro
In vitro ovarian average diameter is slightly less than the in vivo growth of ovary, but the difference was not statistically significant; HE staining of in vitro culture of ovarian tissue pathology and no significant change in the tenth days of incubation of primordial follicles decreased significantly, statistically significant, the number of primary follicles and secondary follicles were no significant difference between.Ki-67 and Caspase3 immunohistochemical staining showed that cultured in vitro ovarian apoptosis increased, with statistical significance, mainly for primordial follicle apoptosis; no significant difference.
2. bisphenol A reduces the number of primordial follicle pools through overactivation of the primordial follicle development
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