本文選題：雞 + 卵巢　； 參考：《華中農(nóng)業(yè)大學(xué)》2017年博士論文

【摘要】：卵巢發(fā)育是一個(gè)受多因子調(diào)控的動態(tài)過程,其發(fā)育狀態(tài)直接影響母雞的產(chǎn)蛋能力,但具體的調(diào)控機(jī)制以及卵泡選擇機(jī)理仍不清楚。顆粒細(xì)胞是最早發(fā)育的卵泡體細(xì)胞,其分化與卵泡選擇過程密切相關(guān)。FOXL2(Forkhead box L2)是哺乳動物卵巢發(fā)育的重要調(diào)控因子。本研究將圍繞FOXL2在雞卵泡顆粒細(xì)胞分化過程中的作用進(jìn)行系統(tǒng)研究。首先使用定量PCR、整體原位雜交和免疫熒光等技術(shù)對FOXL2在雞胚胎性腺及性成熟卵巢中的表達(dá)模式進(jìn)行研究,為探索FOXL2在雞卵巢發(fā)育過程中的作用提供基礎(chǔ);其次離體培養(yǎng)雞等級前(SGCs)和等級卵泡顆粒細(xì)胞(BGCs),并將重組的FOXL2真核表達(dá)載體轉(zhuǎn)染入離體培養(yǎng)的雞卵泡顆粒細(xì)胞中,使用高通量測序技術(shù)檢測FOXL2過表達(dá)后細(xì)胞內(nèi)轉(zhuǎn)錄本的變化情況,并利用生物信息學(xué)技術(shù)分析FOXL2對雞卵泡顆粒細(xì)胞的調(diào)控作用;最后使用雙熒光素酶報(bào)告基因的方法檢測FOXL2對芳香化酶基因(Cytochrome P450 aromatase,P450arom/CYP19)啟動子活性的調(diào)控作用。主要研究結(jié)果如下:(1)在雞胚胎期泌尿生殖系統(tǒng)中,FOXL2主要在雌性性腺中表達(dá),在雄性性腺中始終無明顯表達(dá)。在雌性性腺中,FOXL2在E4.5開始表達(dá),隨著胚齡增加逐漸上調(diào)。在性成熟母雞卵泡中,FOXL2主要在卵泡顆粒細(xì)胞中表達(dá),而膜細(xì)胞中無明顯表達(dá)。FOXL2在小白卵泡和大白卵泡中即有表達(dá),在小黃卵泡顆粒細(xì)胞中開始上調(diào)并在等級卵泡顆粒細(xì)胞中表達(dá)量維持在一個(gè)較高的水平,到F1卵泡顆粒細(xì)胞中表達(dá)量達(dá)到高峰。(2)在離體培養(yǎng)的顆粒細(xì)胞中分別轉(zhuǎn)染空載體(對照組)和FOXL2真核表達(dá)載體后(過表達(dá)組),使用定量PCR檢測FOXL2的表達(dá)量,結(jié)果顯示與對照組相比,過表達(dá)組中FOXL2的表達(dá)量極顯著上調(diào)(SGCs中超過20倍,BGCs中超過6倍)。(3)選擇同一批次處理的SGCs和BGCs(對照組及過表達(dá)組)各2個(gè)進(jìn)行數(shù)字化表達(dá)譜測序,共得到97,148,313條Clean reads,通過與參考基因序列比對注釋全部8個(gè)樣本共獲得14,280個(gè)基因。將樣本按照如下的組進(jìn)行兩兩對比:CTRL-BGCs vs.CTRL-SGCs(命名為Comp.1),FOXL2-SGCs vs.CTRL-SGCs(命名為Comp.2),FOXL2-BGCs vs.CTRL-BGCs(命名為Comp.3)。結(jié)果發(fā)現(xiàn)在Comp.1中有1,575個(gè)差異基因(Differentially expressed genes,DEGs)(885個(gè)上調(diào)基因,690個(gè)下調(diào)基因);在Comp.2中有207個(gè)DEGs(112個(gè)基因被FOXL2激活,95個(gè)基因被抑制);在Comp.3中有201被FOXL2調(diào)控的基因(84個(gè)被激活基因,117個(gè)被抑制基因)。(4)GO注釋分析發(fā)現(xiàn)在Comp.1中超過90%差異表達(dá)基因與細(xì)胞學(xué)過程相關(guān);在Comp.2中DEGs主要與細(xì)胞內(nèi)信號轉(zhuǎn)導(dǎo)過程;在Comp.3中DEGs主要影響細(xì)胞運(yùn)動性以及細(xì)胞外基質(zhì)形成等過程。KEGG通路分析發(fā)現(xiàn)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主要影響“cytokine-cytokine receptor interaction”通路;Comp.3中的DEGs主要影響“focal adhesion”和“ECM-receptor interaction”通路。(5)使用Comp.1和Comp.2的并集基因結(jié)果篩選FOXL2在顆粒細(xì)胞分化過程中作用的候選通路和候選基因。結(jié)果顯示FOXL2可能是通過“focal adhesion”和“cytokine-cytokine receptor interaction”兩條通路調(diào)控雞卵泡顆粒細(xì)胞的分化過程。(6)綜合分析3組數(shù)據(jù)發(fā)現(xiàn)Comp.2和Comp.3的共同基因表達(dá)變化趨勢幾乎均相反。STEM表達(dá)模式結(jié)果顯示當(dāng)FOXL2在等級前顆粒細(xì)胞中過表達(dá)后,基因表達(dá)模式的變化方向與顆粒細(xì)胞分化過程中的模式相同;而當(dāng)FOXL2在等級卵泡顆粒細(xì)胞中過表達(dá)后,其卻可調(diào)控相關(guān)基因表達(dá)量接近未分化時(shí)。(7)FOXL2與芳香化酶表達(dá)不共定位,并且FOXL2不能影響CYP19A1啟動子活性,而類固醇生成因子(Steroidogenic factor 1,SF-1)可以上調(diào)CYP19A1啟動子活性,并呈現(xiàn)劑量依賴的特性。
[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.

【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：S831

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本文編號：1795564