FOXL2对鸡卵泡颗粒细胞分化的作用及机制研究

发布时间：2018-04-24 06:43

本文选题：鸡 + 卵巢　；参考：《华中农业大学》2017年博士论文

【摘要】：卵巢发育是一个受多因子调控的动态过程,其发育状态直接影响母鸡的产蛋能力,但具体的调控机制以及卵泡选择机理仍不清楚。颗粒细胞是最早发育的卵泡体细胞,其分化与卵泡选择过程密切相关。FOXL2(Forkhead box L2)是哺乳动物卵巢发育的重要调控因子。本研究将围绕FOXL2在鸡卵泡颗粒细胞分化过程中的作用进行系统研究。首先使用定量PCR、整体原位杂交和免疫荧光等技术对FOXL2在鸡胚胎性腺及性成熟卵巢中的表达模式进行研究,为探索FOXL2在鸡卵巢发育过程中的作用提供基础;其次离体培养鸡等级前(SGCs)和等级卵泡颗粒细胞(BGCs),并将重组的FOXL2真核表达载体转染入离体培养的鸡卵泡颗粒细胞中,使用高通量测序技术检测FOXL2过表达后细胞内转录本的变化情况,并利用生物信息学技术分析FOXL2对鸡卵泡颗粒细胞的调控作用;最后使用双荧光素酶报告基因的方法检测FOXL2对芳香化酶基因(Cytochrome P450 aromatase,P450arom/CYP19)启动子活性的调控作用。主要研究结果如下:(1)在鸡胚胎期泌尿生殖系统中,FOXL2主要在雌性性腺中表达,在雄性性腺中始终无明显表达。在雌性性腺中,FOXL2在E4.5开始表达,随着胚龄增加逐渐上调。在性成熟母鸡卵泡中,FOXL2主要在卵泡颗粒细胞中表达,而膜细胞中无明显表达。FOXL2在小白卵泡和大白卵泡中即有表达,在小黄卵泡颗粒细胞中开始上调并在等级卵泡颗粒细胞中表达量维持在一个较高的水平,到F1卵泡颗粒细胞中表达量达到高峰。(2)在离体培养的颗粒细胞中分别转染空载体(对照组)和FOXL2真核表达载体后(过表达组),使用定量PCR检测FOXL2的表达量,结果显示与对照组相比,过表达组中FOXL2的表达量极显著上调(SGCs中超过20倍,BGCs中超过6倍)。(3)选择同一批次处理的SGCs和BGCs(对照组及过表达组)各2个进行数字化表达谱测序,共得到97,148,313条Clean reads,通过与参考基因序列比对注释全部8个样本共获得14,280个基因。将样本按照如下的组进行两两对比:CTRL-BGCs vs.CTRL-SGCs(命名为Comp.1),FOXL2-SGCs vs.CTRL-SGCs(命名为Comp.2),FOXL2-BGCs vs.CTRL-BGCs(命名为Comp.3)。结果发现在Comp.1中有1,575个差异基因(Differentially expressed genes,DEGs)(885个上调基因,690个下调基因);在Comp.2中有207个DEGs(112个基因被FOXL2激活,95个基因被抑制);在Comp.3中有201被FOXL2调控的基因(84个被激活基因,117个被抑制基因)。(4)GO注释分析发现在Comp.1中超过90%差异表达基因与细胞学过程相关;在Comp.2中DEGs主要与细胞内信号转导过程;在Comp.3中DEGs主要影响细胞运动性以及细胞外基质形成等过程。KEGG通路分析发现Comp.1中的DEGs主要富集在“focal adhesion”,“cell cycle”,以及“ECM-receptor interaction”通路中;Comp.2中的DEGs主要影响“cytokine-cytokine receptor interaction”通路;Comp.3中的DEGs主要影响“focal adhesion”和“ECM-receptor interaction”通路。(5)使用Comp.1和Comp.2的并集基因结果筛选FOXL2在颗粒细胞分化过程中作用的候选通路和候选基因。结果显示FOXL2可能是通过“focal adhesion”和“cytokine-cytokine receptor interaction”两条通路调控鸡卵泡颗粒细胞的分化过程。(6)综合分析3组数据发现Comp.2和Comp.3的共同基因表达变化趋势几乎均相反。STEM表达模式结果显示当FOXL2在等级前颗粒细胞中过表达后,基因表达模式的变化方向与颗粒细胞分化过程中的模式相同;而当FOXL2在等级卵泡颗粒细胞中过表达后,其却可调控相关基因表达量接近未分化时。(7)FOXL2与芳香化酶表达不共定位,并且FOXL2不能影响CYP19A1启动子活性,而类固醇生成因子(Steroidogenic factor 1,SF-1)可以上调CYP19A1启动子活性,并呈现剂量依赖的特性。
[Abstract]:Ovarian development is a dynamic process controlled by multiple factors. Its developmental state directly affects the laying ability of hens, but the specific regulation mechanism and the mechanism of follicle selection are still unclear. Granulosa cells are the earliest developed follicle cells, and their differentiation is closely related to the follicle selection process.FOXL2 (Forkhead box L2) is the mammalian ovary. This study will systematically study the role of FOXL2 in the differentiation of chicken follicle granulosa cells. First, quantitative PCR, overall in situ hybridization and immunofluorescence techniques are used to study the expression patterns of FOXL2 in the gonadal and sexually mature ovaries of chicken embryos, in order to explore the development of FOXL2 in the development of chicken ovary. The function provided the basis; secondly, in vitro culture of chicken grade (SGCs) and grade follicle granulosa cells (BGCs), the recombinant FOXL2 eukaryotic expression vector was transfected into the cultured chicken follicle granulosa cells. High throughput sequencing technique was used to detect the changes in the intracellular transcript of FOXL2 overexpression, and the bioinformatics analysis was used. The regulation of FOXL2 on the granulosa cells of chicken follicles; and finally using the method of double luciferase reporter gene to detect the regulation of FOXL2 on the activity of the Cytochrome P450 aromatase (P450arom/CYP19) promoter. The main results are as follows: (1) in the genitourinary system of the embryonic stage of the chicken, FOXL2 is mainly expressed in the female gonadal gland. There was no obvious expression in the male gonadal gland. In the female gonadal gland, FOXL2 began to express in E4.5, and gradually increased with the increase of embryo age. In the follicles of sexually mature hens, FOXL2 was mainly expressed in follicle granulosa cells, but there was no obvious expression of.FOXL2 in the white follicle and white follicle in the membrane cells, and in the small yellow follicle granulosa cells. The expression in the grade follicle granulosa cells was maintained at a high level and reached a peak in the F1 follicle granulosa cells. (2) after transfecting empty carriers (control group) and FOXL2 eukaryotic expression vector in the isolated cultured granulosa cells (over expressed group), quantitative PCR was used to detect the expression of FOXL2. The results showed that Compared with the control group, the expression of FOXL2 in the overexpressed group was significantly up-regulated (more than 20 times in SGCs, more than 6 times in BGCs). (3) a total of 2 SGCs and BGCs (control and overexpressed groups) were sequenced in the same batch, and 97148313 Clean reads were obtained, and all 8 samples were annotated by comparison with the reference gene sequence. 14280 genes were obtained. The samples were compared in the following 22 groups: CTRL-BGCs vs.CTRL-SGCs (named Comp.1), FOXL2-SGCs vs.CTRL-SGCs (named Comp.2), FOXL2-BGCs vs.CTRL-BGCs (named Comp.3). The results found that there were 1575 differential genes (Differentially expressed) in Comp.1 (885 up-regulated genes, 690) There were 207 DEGs in Comp.2 (112 genes were activated by FOXL2 and 95 genes were suppressed); 201 in FOXL2 were regulated by FOXL2 (84 activated genes, 117 suppressed genes). (4) GO annotation analysis found that more than 90% differential tables were related to the cytological process in Comp.1; DEGs was mainly in Comp.2 in the cell The process of signal transduction; in Comp.3, DEGs mainly affects the activity of cell and the formation of extracellular matrix. It is found that the DEGs in Comp.1 is mainly enriched in "focal adhesion", "cell cycle" and "ECM-receptor interaction". The action pathway; the DEGs in Comp.3 mainly affects the "focal adhesion" and "ECM-receptor interaction" pathway. (5) the candidate pathways and candidate genes of FOXL2 in the process of granulosa cell differentiation are screened using the aggregation gene results of Comp.1 and Comp.2. Ine receptor interaction "two pathways regulate the differentiation process of chicken follicle granulosa cells. (6) comprehensive analysis of 3 groups of data found that the common gene expression changes of Comp.2 and Comp.3 almost all the opposite.STEM expression pattern results show that when FOXL2 is overexpressed in the pre grade granulosa cells, the direction of the gene expression pattern and the particle size are fine. The pattern of cell differentiation is the same, but when FOXL2 is overexpressed in the grade follicle granulosa cells, it can regulate the expression of related genes close to undifferentiated. (7) the expression of FOXL2 and aromatase is not Co located, and FOXL2 can not affect the activity of CYP19A1 promoter, and the steroid generating factor (Steroidogenic factor 1, SF-1) can up regulate the CYP. 19A1 promoter activity showed a dose-dependent nature.

【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S831

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