本文關鍵詞：大鼠腔前卵泡體外三維培養(yǎng)系統(tǒng)的建立及其在雌性生殖毒性研究中的應用　出處：《第二軍醫(yī)大學》2012年碩士論文　論文類型：學位論文

  更多相關文章： 卵泡 三維 海藻酸鈉 卵母細胞成熟 體外培養(yǎng) BPA

【摘要】：目前雌性生殖毒性評價方法主要采用整體動物試驗，存在的主要問題是所需動物數多，不符合3R原則；實驗周期長、所需受試樣品量多，難以滿足化學品快速發(fā)展的需求；所提供的毒作用機制等信息有限，不足以提供安全性和危險度評定的需求，，也不符合現代毒理學發(fā)展的要求。為此，近幾年國內外已經建立了多種體外雌性生殖毒性試驗方法，包括卵巢器官和卵巢皮質薄片培養(yǎng)方法和大鼠顆粒細胞和黃體細胞體外毒性試驗法。但這些體外方法無法對卵泡發(fā)育和卵子生成做出客觀評價，而這恰恰是雌性生殖毒性研究的關鍵所在。因此，迫切需要發(fā)展既能快速檢測雌性生殖毒性又能更好地研究其作用機制的新方法。近年新發(fā)展的體外卵泡培養(yǎng)方法，能對卵巢的主要功能即卵泡發(fā)育、激素生成和卵子發(fā)生進行動態(tài)觀察，可在卵泡不同發(fā)育階段對卵泡每一組分進行分析，對卵泡發(fā)育,卵子發(fā)生和激素調控等生殖生物學機制和生殖毒作用機制進行深入研究，因而受到生殖生物學、臨床醫(yī)學和生殖毒理學等領域的關注。 我室萬旭英等率先建立了大鼠腔前卵泡體外二維培養(yǎng)模型，但未來體外毒性測試方法的發(fā)展趨勢是發(fā)展基于三維培養(yǎng)模型的方法，目前國外已有小鼠腔前卵泡三維培養(yǎng)模型的報道，但大鼠僅有短期三維培養(yǎng)的報道，尚無大鼠腔前卵泡體外三維完整培養(yǎng)模型的研究報道。 本研究擬在以往研究基礎上，建立一個能支持大鼠腔前卵泡生長并產生成熟卵母細胞又適用于毒性研究的體外三維培養(yǎng)系統(tǒng)；然后用雌性生殖毒物進行驗證研究。目的是為建立一個既可對雌性生殖毒物進行篩選或鑒定，又可對生殖毒作用機制進行研究的體外毒性試驗系統(tǒng)提供依據。 一、大鼠腔前卵泡體外三維培養(yǎng)系統(tǒng)的建立 目的：建立一個支持大鼠腔前卵泡生長并且能夠產生成熟卵母細胞的體外培養(yǎng)系統(tǒng)。方法：從12-14天齡的雌性SD大鼠卵巢中機械性分離腔前卵泡（140-170μm），然后在三維的海藻酸鈉和二維的微孔板中連續(xù)培養(yǎng)10天。三維培養(yǎng)效果通過卵泡發(fā)育的形態(tài)學、激素分泌，排卵率以及卵母細胞成熟率等進行評價,并與二維培養(yǎng)結果進行比較。結果：在三維培養(yǎng)期間卵泡一直保持著三維的結構，而在二維培養(yǎng)系統(tǒng)中培養(yǎng)兩天后卵泡膜細胞開始貼壁生長，在接下來的培養(yǎng)過程中大部分卵泡的卵泡膜細胞向四周擴散，顆粒細胞突破基底膜。三維培養(yǎng)系統(tǒng)中卵泡的存活率、有腔形成率、COCs排出率以及PB形成率均高于二維培養(yǎng)系統(tǒng)，分別為95.7vs.90.7%（P0.05）、72.1vs.33.6%（P0.05）、79.3vs.39.8%（P0.05），57.1vs.31.9%（P0.05）。在兩種培養(yǎng)系統(tǒng)中，從腔前卵泡到卵母細胞成熟以及第一極體的形成，形態(tài)學觀察顯示大鼠腔前卵泡體外發(fā)育特征和體內相似。雌二醇和孕酮的分泌模式與卵泡的形態(tài)學變化以及分化情況相一致。結論：本研究建立的大鼠腔前卵泡體外三維培養(yǎng)系統(tǒng)是成功的。 二、雙酚-A對大鼠卵泡體外生長發(fā)育和卵母細胞成熟的影響 目的：觀察雙酚-A（BPA）對大鼠卵泡體外生長發(fā)育及卵母細胞成熟的影響，驗證該系統(tǒng)用于生殖毒性研究的可行性。方法：從12-14天齡的雌性SD大鼠卵巢中機械性分離腔前卵泡（140-170μm），隨機分為3個處理組（50~150μM）及一個對照組，然后在三維的海藻酸鈉中連續(xù)培養(yǎng)10天。結果通過卵泡發(fā)育的形態(tài)學、激素分泌，排卵率以及卵母細胞成熟率等指標進行評價。結果：對照組卵泡在連續(xù)的10天培養(yǎng)過程當中，絕大多數都經歷了腔前卵泡、有腔卵泡以及成熟卵泡階段。與對照組相比，隨著劑量的增加卵泡存活率、有腔形成率， COCs排出率以及PB率均降低，100μM及以上劑量下與對照組相比差異有統(tǒng)計學意義（P0.05）。處理組中，卵泡膜細胞和顆粒細胞的增殖受到不同程度的抑制，100μM及以上劑量下與對照組相比差異有統(tǒng)計學意義（P0.05）。雌二醇和孕酮的分泌量隨著劑量的增加而減少，與對照組相比差異有統(tǒng)計學意義（P0.05）。結論：本研究劑量下BPA抑制卵泡的生長和卵母細胞的成熟；初步驗證說明大鼠卵泡體外長期三維培養(yǎng)模型適用于毒性研究。 上述結果顯示，本研究建立的三維培養(yǎng)系統(tǒng)能夠支持大鼠腔前卵泡生長并產生成熟卵母細胞，而且在卵泡存活率、有腔形成率、COCs排出率以及PB形成率等方面均優(yōu)于二維培養(yǎng)系統(tǒng)；雌性生殖毒物雙酚-A的研究表明，該系統(tǒng)適用于毒理學研究。表明，本研究成功建立的大鼠腔前卵泡體外三維培養(yǎng)模型具有發(fā)展成為既能對卵巢毒物進行快速評價又能研究雌性生殖毒作用機制新方法的應用前景。
[Abstract]:The evaluation of female reproductive toxicity mainly by animal experiment, the main problems are required for animal number, does not comply with 3R principles; the long period required by the sample volume, it is difficult to meet the demand of the rapid development of chemical; provided the toxic mechanism of information is limited, not enough to provide safety and danger the assessment of needs, does not conform to the requirements of the development of modern toxicology. Therefore, in recent years at home and abroad have established test methods for female reproductive toxicity in vitro, including ovarian organ and ovarian cortex slice culture method and in vitro rat granulosa cells and luteal cells in vitro toxicity test method. But these methods cannot on follicular development and oocyte generation make objective evaluation, and this is precisely the key to female reproductive toxicity research. Therefore, the urgent need for the development of both the rapid detection of female reproductive toxicity and more A new method to study the mechanism of action. In recent years the development of in vitro follicle culture method, the main function of the energy of ovarian follicular development, hormone production and oogenesis were observed dynamically in the follicles at different stages of follicles of each component were analyzed on follicular development, in-depth study of oogenesis and hormone regulation of reproductive biology and reproductive toxicity mechanism, and thus have reproductive biology, clinical medicine and reproductive toxicology field attention.
My room Wan Xuying took the lead in the establishment of rat preantral follicles in vitro culture of two-dimensional model, but the future development trend of in vitro toxicity test method is the development method of three-dimensional culture model based on the reported abroad preantral follicle culture model, but only a short period of rat cultured rat preantral is reported full three-dimensional culture of follicles in vitro model reported.
This study based on previous studies, the establishment of a support rat preantral follicle and mature oocytes and suitable for toxicity study in three-dimensional culture system; and then use the female reproductive toxicants to verify the research. The purpose is to establish a which can be used for screening or identification of female reproductive toxicants. Provide the basis for the test system can be used to study the in vitro toxicity and reproductive toxicity mechanism.
Establishment of a three-dimensional culture system of preantral follicles in vitro in rats
Objective: to establish a rat preantral follicle and can produce mature oocytes in vitro culture system. Methods: from isolated preantral follicles mechanical 12-14 day old female SD rat ovary (140-170 m), then the microplate in three-dimensional alginate and two-dimensional continuous culture for 10 days the effect of three-dimensional culture. The morphology, follicular growth hormone secretion, evaluate the ovulation rate and oocyte maturation rate, and compared with the results of two-dimensional culture. Results: in the three-dimensional culture during follicle has maintained a three-dimensional structure, and in the two-dimensional culture became adherent after two days of culture theca cell system. Spread around in most follicular theca cells in the process of cultivating the granular cells through the basement membrane. The three-dimensional culture system of follicle survival rate, cavity formation rate, COCs removal rate and PB The formation rate was higher than that of two-dimensional culture system were 95.7vs.90.7% (P0.05), 72.1vs.33.6% (P0.05), 79.3vs.39.8% (P0.05), 57.1vs.31.9% (P0.05). In two culture systems, from follicle to oocyte maturation and the first polar body cavity is formed before the morphological observation showed that rat preantral follicle development in vitro characteristics and in vivo. Similar morphologic changes consistent with secretion and follicular estradiol and progesterone and differentiation of rat. Conclusion: This study established the follicular cavity in three-dimensional culture system is successful.
Two, the effect of bisphenol -A on the growth and development of oocyte in vitro and oocyte maturation in rats
Objective: To observe the effects of bisphenol -A (BPA) on rat follicle growth and oocyte maturation, verify the system feasibility study for the reproductive toxicity. Methods: from isolated preantral follicles mechanical 12-14 day old female SD rat ovary (140-170 m), were randomly divided into 3 treatments group (50~150 M) and a control group, and then the three-dimensional alginate in continuous culture for 10 days. The morphology, follicular growth hormone secretion, evaluate the ovulation rate and oocyte maturation rate. Results: the control group of follicles cultured in 10 consecutive days process, the vast majority of experience the preantral follicles, antral follicles and mature follicles. Compared with the control group, with the increase of the dosage of follicle survival rate, cavity formation rate, COCs removal rate and PB rate were reduced, and more than 100 M dose compared with the control group had statistical difference Meaning (P0.05). Treatment group, theca cells and granulosa cell proliferation was inhibited in different degrees, and over 100 M dose compared with the control group the difference was statistically significant (P0.05). The secretion of estradiol and progesterone decreased with the increase of dose, compared with the control group the difference was statistically significant (P0.05). Conclusion: the dose of BPA inhibits the growth and maturation of oocytes of follicles; to demonstrate the preliminary toxicity study for a long time and three-dimensional culture of rat follicle in vitro model.
The results show that the three-dimensional culture system established in this study can support rat preantral follicle and mature oocytes, and the survival rate in the follicular cavity formation rate, COCs and PB excretion rate was significantly higher than that of the formation of two-dimensional culture system; Study on female reproductive toxicants bisphenol -A showed that in toxicology research the application of the system. This study showed that the rat cavity successfully established three-dimensional model has become the development of follicle culture in vitro can be evaluated quickly and application prospect to study ovarian toxicants new method of female reproductive toxicity.

【學位授予單位】：第二軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R114

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