构建小鼠卵巢早衰模型研究p204在卵泡发育中的作用

发布时间：2018-01-31 06:34

本文关键词： p204 卵巢早衰 D-半乳糖小鼠模型　出处：《山东大学》2017年硕士论文　论文类型：学位论文

【摘要】：干扰素(IFN)诱导蛋白在干扰素生物学功能的发挥中起着至关重要的作用,p204是干扰素诱导蛋白p200家族(IFI200)鼠类蛋白成员,该蛋白能与多种蛋白相互作用,并通过这些相互作用发挥抑制细胞增殖、阻断细胞周期、促进细胞分化及感应外源双链DNA入侵等重要作用。本论文用D-半乳糖注射法对遗传背景相同的全身性p204基因敲除型(KO)小鼠和野生型(WT)小鼠进行卵巢早衰模型的构建,观察小鼠造模过程中的健康状况。造模八周后组织切片染色观察卵巢卵泡的发育的状况。采用酶联免疫吸附法检测血清睾酮(T)、雌二醇(E2)、促卵泡激素(FSH)、黄体生成素(HL)水平。采用real-timePCR技术对卵巢组织c-myc、Bcl-2、Bax、CYP17A1、CYP19A1等基因表达水平进行检测与分析。采用免疫组化法检测Bcl-2、CYP17A1、CYP19A1蛋白在小鼠卵巢组织的表达和分布情况。实验结果表明,在造模过程中,KO小鼠的体重及卵巢重量均显著下降(p0.01),而WT小鼠变化不显著,说明p204敲除使小鼠对D-半乳糖代谢紊乱导致的生理功能变化的影响更大。通过对卵巢组织的切片及染色观察发现KO小鼠卵巢闭锁卵泡和黄体增多,颗粒细胞排列稀疏,说明p204敲除对小鼠卵巢卵泡的发育产生了一定影响。对小鼠血清激素水平检测发现KO小鼠睾酮(T)、促卵泡激素(FSH)、黄体生成素(LH)水平均显著升高(p0.01)。说明p204基因敲除后使小鼠体内的激素水平发生改变,导致卵泡发育功能下降,致使卵巢损伤。对小鼠卵巢相关基因检测中,发现KO小鼠凋亡相关基因bax、原癌基因c-myc表达水平均显著升高(p0.01),说明p204敲除对小鼠卵巢颗粒细胞凋亡产生影响,加速了卵巢早衰。还发现KO小鼠中性激素合成相关基因CYP17A1表达水平显著升高(p0.01),CYP19A1基因表达水平均显著降低(p0.01),说明p204敲除使小鼠体内性激素尤其是雄激素表达水平发生改变,导致小鼠卵泡发育发生障碍,形成卵巢早衰。免疫组化结果显示缺失p204导致小鼠卵巢内Bcl-2、CYP17A1、CYP19A1蛋白的表达和分布发生改变,加速卵巢早衰形成。以上结果表明p204在小鼠卵巢卵泡发育中可能通过调控颗粒细胞增殖和凋亡的过程从而抵制卵巢早衰的形成。本论文为初步探究p204在小鼠POF卵泡发育中的作用,为进一步阐明卵巢早衰卵泡发育障碍的发病机制和预防早衰奠定基础,具有重要的理论意义和临床价值。本研究的创新点在于:1)使用D-半乳糖注射法对C57品系p204基因全身性敲除小鼠构建卵巢早衰模型。2)发现缺失p204对小鼠卵巢组织产生损伤加速卵巢早衰。
[Abstract]:IFN- induced protein plays an important role in the biological function of interferon. P204 is a member of IFI200, a family of interferon inducible proteins (p200). The protein can interact with a variety of proteins, and through these interactions play a role in inhibiting cell proliferation, blocking cell cycle. Promoting cell differentiation and induction of exogenous double-stranded DNA invasion. In this study, we used Dgalactose injection method to study the genetic background of the mice with the same genetic background of p204 gene knockout type KOO and the wild type of p204. The model of premature ovarian failure was constructed in mice. The development of ovarian follicles was observed by histological staining eight weeks later. Serum testosterone (TX) and estradiol (E2) were detected by enzyme-linked immunosorbent assay (Elisa). The levels of follicle stimulating hormone (FSH) and luteinizing hormone (LH) were determined by real-timePCR technique. The expression level of CYP19A1 and other genes were detected and analyzed. Bcl-2CYP 17A1 was detected by immunohistochemical method. The expression and distribution of CYP19A1 protein in mouse ovary. The results showed that the body weight and ovarian weight of KO mice decreased significantly (p0.01). The change of WT mice was not significant. The results showed that p204 knockout had a greater effect on the physiological function of mice caused by D-galactose metabolism disorder. The ovarian atresia follicles and luteal bodies of KO mice were found to be increased by the observation of sections and staining of ovarian tissue. The sparse arrangement of granulosa cells indicated that p204 knockout had a certain effect on the development of ovarian follicles in mice. The level of luteinizing hormone (LH) increased significantly (P 0.01), which indicated that the hormone level of mice was changed after p204 gene knockout, which resulted in the decline of follicle development function. The expression level of apoptosis-related gene baxand proto-oncogene c-myc in KO mice were significantly increased (P 0.01). The results showed that p204 knockout had an effect on the apoptosis of granulosa cells in mouse ovary. It was also found that the CYP17A1 expression level of sex hormone synthesis-related gene in KO mice was significantly increased (p 0.01). The expression level of CYP19A1 gene decreased significantly, indicating that p204 knockout could change the expression of sex hormone, especially androgen, and lead to follicular dysfunction in mice. The expression and distribution of CYP19A1 protein in mouse ovary were changed by the deletion of p204. The above results suggest that p204 may resist the formation of premature ovarian failure by regulating the process of granulosa cell proliferation and apoptosis. (4) role in the development of mouse POF follicles. In order to further elucidate the pathogenesis of ovarian premature failure follicular dysplasia and prevent premature failure lay a foundation. This study has important theoretical significance and clinical value. The innovation of this study lies in the use of Dgalactose injection method to construct premature ovarian failure model of C57 strain p204 gene knockout mice. It was found that the absence of p204 caused damage to ovarian tissue and accelerated premature ovarian failure in mice.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R711.75;R-332

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