大鼠腔前卵泡体外三维培养系统的建立及其在雌性生殖毒性研究中的应用

发布时间：2018-01-16 03:23

本文关键词：大鼠腔前卵泡体外三维培养系统的建立及其在雌性生殖毒性研究中的应用　出处：《第二军医大学》2012年硕士论文　论文类型：学位论文

【摘要】：目前雌性生殖毒性评价方法主要采用整体动物试验，存在的主要问题是所需动物数多，不符合3R原则；实验周期长、所需受试样品量多，难以满足化学品快速发展的需求；所提供的毒作用机制等信息有限，不足以提供安全性和危险度评定的需求，，也不符合现代毒理学发展的要求。为此，近几年国内外已经建立了多种体外雌性生殖毒性试验方法，包括卵巢器官和卵巢皮质薄片培养方法和大鼠颗粒细胞和黄体细胞体外毒性试验法。但这些体外方法无法对卵泡发育和卵子生成做出客观评价，而这恰恰是雌性生殖毒性研究的关键所在。因此，迫切需要发展既能快速检测雌性生殖毒性又能更好地研究其作用机制的新方法。近年新发展的体外卵泡培养方法，能对卵巢的主要功能即卵泡发育、激素生成和卵子发生进行动态观察，可在卵泡不同发育阶段对卵泡每一组分进行分析，对卵泡发育,卵子发生和激素调控等生殖生物学机制和生殖毒作用机制进行深入研究，因而受到生殖生物学、临床医学和生殖毒理学等领域的关注。我室万旭英等率先建立了大鼠腔前卵泡体外二维培养模型，但未来体外毒性测试方法的发展趋势是发展基于三维培养模型的方法，目前国外已有小鼠腔前卵泡三维培养模型的报道，但大鼠仅有短期三维培养的报道，尚无大鼠腔前卵泡体外三维完整培养模型的研究报道。本研究拟在以往研究基础上，建立一个能支持大鼠腔前卵泡生长并产生成熟卵母细胞又适用于毒性研究的体外三维培养系统；然后用雌性生殖毒物进行验证研究。目的是为建立一个既可对雌性生殖毒物进行筛选或鉴定，又可对生殖毒作用机制进行研究的体外毒性试验系统提供依据。一、大鼠腔前卵泡体外三维培养系统的建立目的：建立一个支持大鼠腔前卵泡生长并且能够产生成熟卵母细胞的体外培养系统。方法：从12-14天龄的雌性SD大鼠卵巢中机械性分离腔前卵泡（140-170μm），然后在三维的海藻酸钠和二维的微孔板中连续培养10天。三维培养效果通过卵泡发育的形态学、激素分泌，排卵率以及卵母细胞成熟率等进行评价,并与二维培养结果进行比较。结果：在三维培养期间卵泡一直保持着三维的结构，而在二维培养系统中培养两天后卵泡膜细胞开始贴壁生长，在接下来的培养过程中大部分卵泡的卵泡膜细胞向四周扩散，颗粒细胞突破基底膜。三维培养系统中卵泡的存活率、有腔形成率、COCs排出率以及PB形成率均高于二维培养系统，分别为95.7vs.90.7%（P0.05）、72.1vs.33.6%（P0.05）、79.3vs.39.8%（P0.05），57.1vs.31.9%（P0.05）。在两种培养系统中，从腔前卵泡到卵母细胞成熟以及第一极体的形成，形态学观察显示大鼠腔前卵泡体外发育特征和体内相似。雌二醇和孕酮的分泌模式与卵泡的形态学变化以及分化情况相一致。结论：本研究建立的大鼠腔前卵泡体外三维培养系统是成功的。二、双酚-A对大鼠卵泡体外生长发育和卵母细胞成熟的影响目的：观察双酚-A（BPA）对大鼠卵泡体外生长发育及卵母细胞成熟的影响，验证该系统用于生殖毒性研究的可行性。方法：从12-14天龄的雌性SD大鼠卵巢中机械性分离腔前卵泡（140-170μm），随机分为3个处理组（50~150μM）及一个对照组，然后在三维的海藻酸钠中连续培养10天。结果通过卵泡发育的形态学、激素分泌，排卵率以及卵母细胞成熟率等指标进行评价。结果：对照组卵泡在连续的10天培养过程当中，绝大多数都经历了腔前卵泡、有腔卵泡以及成熟卵泡阶段。与对照组相比，随着剂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及以上剂量下与对照组相比差异有统计学意义（P0.05）。处理组中，卵泡膜细胞和颗粒细胞的增殖受到不同程度的抑制，100μM及以上剂量下与对照组相比差异有统计学意义（P0.05）。雌二醇和孕酮的分泌量随着剂量的增加而减少，与对照组相比差异有统计学意义（P0.05）。结论：本研究剂量下BPA抑制卵泡的生长和卵母细胞的成熟；初步验证说明大鼠卵泡体外长期三维培养模型适用于毒性研究。上述结果显示，本研究建立的三维培养系统能够支持大鼠腔前卵泡生长并产生成熟卵母细胞，而且在卵泡存活率、有腔形成率、COCs排出率以及PB形成率等方面均优于二维培养系统；雌性生殖毒物双酚-A的研究表明，该系统适用于毒理学研究。表明，本研究成功建立的大鼠腔前卵泡体外三维培养模型具有发展成为既能对卵巢毒物进行快速评价又能研究雌性生殖毒作用机制新方法的应用前景。
[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.

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R114

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