本文選題：雙酚A　切入點：卵巢培養(yǎng)　出處：《第二軍醫(yī)大學》2014年碩士論文　論文類型：學位論文

【摘要】：[研究背景及目的] 內(nèi)分泌干擾物(Endocrine disruptor chemicals, EDCs)是一種外源性的化合物,能影響人類和動物體內(nèi)的內(nèi)分泌系統(tǒng),進而影響相應器官的功能,甚至是下一代的健康。因其在自然界的廣泛存在,以及對健康的影響,近幾年,EDCs受到廣泛的關(guān)注。 雙酚A(Bisphenol A, BPA)是目前使用量最大的內(nèi)分泌干擾物,在許多常見消費類產(chǎn)品中存在,包括塑料(為一種聚合物,即聚碳酸酯塑料)、聚氯乙烯(PVC)、食品包裝、牙科密封劑等。人類可以通過飲食、空氣吸入以及皮膚接觸吸收BPA。90%的抽樣人群尿液中可檢測出雙酚A,50%的人群血液中可檢測到,美國、德國、中國以及一些其它亞洲國家都得到相似的結(jié)果。更引起人們注意的是,即使很小劑量的BPA都可引起人類的不良反應,因此,多個國家均頒布法律條例來嚴格控制BPA的用量。然而,BPA對人類健康的影響已然存在,只有明確其所致疾病以及致病機制,才能更好的預防和治療環(huán)境相關(guān)疾病。 大量研究顯示,雙酚A可導致多囊卵巢綜合征(Polycystic Ovary Syndrome, PCOS)、不孕癥、流產(chǎn)、代謝綜合征等疾病。雙酚A對人類的健康影響是復雜的、全身的、涉及多個器官,特別是生殖系統(tǒng)。無論是女性還是男性,其生殖系統(tǒng)都容易受到雙酚A的影響。雙酚A能降低男性精子的質(zhì)量、引起精原細胞的凋亡；而對于女性卵巢而言,雙酚A不僅影響發(fā)育卵泡的成熟與排出,還干擾顆粒細胞、卵泡膜細胞中性激素合成、分泌及代謝相關(guān)的酶活性,然而,雙酚A對始基卵泡的研究還很少。 始基卵泡是卵泡發(fā)育的起始階段,也是女性生育功能的儲備池。既往對始基卵泡的相關(guān)研究很少,近幾年,人們逐漸認識了始基卵泡發(fā)育與閉鎖的過程,其中PTEN/PI3K途徑對于始基卵泡池的維持其重要的調(diào)節(jié)作用。出生后,人類的始基卵泡不再增加,而是逐漸減少。始基卵泡的減少速度是恒定的,無論是減少過快或者減少過慢,都有可能影響后續(xù)卵泡的發(fā)育以及整體的卵巢功能。許多因素可影響始基卵泡的數(shù)量,特別是環(huán)境化合物,如VCD(4-vinylcyclohexene diepoxide)、二甲基苯芘蒽(7,12-dimethylbenz [a] anthracene,DMBA)等。已有研究顯示,新生鼠注射雙酚A能顯著減少始基卵泡的數(shù)量而增加初級卵泡的數(shù)量,在新生羊中也觀察到相同結(jié)果。然而,卵巢中卵泡生長的異質(zhì)性明顯,且卵泡細胞內(nèi)的分子改變受卵巢取出時間的影響較大。因此,本課題采用的是新生小鼠整體卵巢體外培養(yǎng)體系,可以控制外界環(huán)境對卵泡的生長,使得卵巢發(fā)育同步化,減少個體差異,可以作為研究卵泡生長與凋亡的良好載體。本研究將利用體外卵巢培養(yǎng)體系,進一步驗證雙酚A對始基卵泡的影響,同時探索雙酚A作用于始基卵泡的機制。 [研究方法] 1.體外新生小鼠卵巢組織培養(yǎng)體系的建立及其對始基卵泡生長的影響 取新生4天C57BL/6小鼠整體卵巢組織,進行體外培養(yǎng),并與體內(nèi)同等時間卵巢生長相比較。光鏡下計量卵巢的平均直徑；通過HE染色觀察培養(yǎng)0天、5天、10天卵巢組織的病理學改變并計數(shù)各級卵泡；Ki-67及caspase3免疫組化分析體外培養(yǎng)卵巢卵泡的凋亡與生長情況。 2.觀察雙酚A對始基卵泡生長的影響 將卵巢組織隨機分為4組,分別為：1、對照組(DMSO);2、低濃度雙酚A組(0.1μM)；3、中等濃度雙酚A組(1μM)；4、高濃度雙酚A組(10μM)。通過HE染色比較各組卵巢的病理改變,并計數(shù)各級卵泡；通過Ki-67及caspase3免疫組化分析各組卵泡的生長與凋亡狀況。 3.探索雙酚A對卵巢組織PI3K/Akt途徑的影響 檢測4組卵巢組織中PI3K/Akt途徑上關(guān)鍵物質(zhì)-PI3K、Akt的表達；用Realtime-PCR檢測mRNA水平上的表達；用Western-blot檢測定量蛋白水平的表達。以PI3K抑制劑阻斷PI3K/Akt途徑后,HE染色計數(shù)初級卵泡數(shù)量。 4.統(tǒng)計學處理 各數(shù)據(jù)均使用SPSS20.0進行統(tǒng)計分析,并用GraphPad Prism5.0軟件制作統(tǒng)計圖表。每組數(shù)值以平均值士標準差表示,計數(shù)資料及方差不齊的資料采用非參數(shù)秩和檢驗(Kruskal-Wallis)進行分析總體差異,隨后將秩和檢驗形成的新的秩變量作單因素方差分析及Dunnett法進行組間比較。其余計量資料均采用one-way ANOVA分析及Dunnett法或Turkey HSD法進行組間比較。P0.05認為差異有統(tǒng)計學意義。 [研究結(jié)果] 1.體外無血清培養(yǎng)條件下的始基卵泡生長與體內(nèi)生長相似 體外培養(yǎng)的卵巢平均直徑略小于體內(nèi)生長卵巢,但差異無統(tǒng)計學意義；HE染色見體外培養(yǎng)卵巢組織病理學無明顯改變,于培養(yǎng)第10天始基卵泡減少明顯,具統(tǒng)計學意義,初級卵泡與次級卵泡數(shù)量均無明顯差異。Ki-67及caspase3免疫組化提示體外培養(yǎng)卵巢凋亡增加,有統(tǒng)計學意義,主要為始基卵泡凋亡；卵泡生長無明顯差異。 2.雙酚A通過過度激活始基卵泡發(fā)育減少始基卵泡池數(shù)量 各濃度組雙酚A與對照組相比,HE染色未見明顯病理學改變,1μM、10μM雙酚A均明顯下降,初級卵泡數(shù)量增加,兩者差異均有統(tǒng)計學意義。而0.1μ M雙酚A組始基卵泡數(shù)量減少與初級卵泡數(shù)量增加差異無統(tǒng)計學意義。各組卵巢次級卵泡數(shù)與卵泡總數(shù)差異無統(tǒng)計學意義。Ki-67及caspase3免疫組化提示各濃度組雙酚A均顯著增加卵泡Ki-67表達,減少caspase3表達,提示卵泡生長增加而凋亡減少。 3.雙酚A通過激活PI3K/Akt途徑促進始基卵泡發(fā)育 各濃度雙酚A組顯著增加PI3K的mRNA和蛋白表達,以及磷酸化Akt的蛋白表達。應用PI3K抑制劑阻斷PI3K/Akt途徑,對照組與雙酚A組的初級卵泡數(shù)量均明顯下降,且含有PI3K抑制劑的對照組與雙酚A組兩者差異無統(tǒng)計學意義。 [研究結(jié)論] 體外小鼠卵巢整體組織培養(yǎng)體系中始基卵泡發(fā)育速度與小鼠體內(nèi)始基卵泡發(fā)育相似,是研究影響始基卵泡閉鎖與發(fā)育的良好載體；雙酚A可顯著減少體外培養(yǎng)的始基卵泡數(shù)量,增加初級卵泡的數(shù)量。而這種作用是雙酚A過度激活始基卵泡發(fā)育、減少始基卵泡凋亡的共同結(jié)果；雙酚A主要通過促進PI3K/Akt途徑表達而發(fā)揮促進始基卵泡發(fā)育的作用。然而,雙酚A如何作用于PI3K/Akt途徑,需要進一步探索研究。
[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
鍚勬祿搴︾粍鍙岄厷A涓庡鐓х粍鐩告瘮,HE鏌撹壊鏈鏄庢樉鐥呯悊瀛︽敼鍙，

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