【摘要】：基質(zhì)金屬蛋白酶(MMPs)在卵泡發(fā)生、發(fā)育、排卵和黃體形成等生物過(guò)程中發(fā)揮著重要的功能。TIMP3作為MMPs的抑制劑可能參與卵泡周期中各生命活動(dòng)間的相互協(xié)調(diào),而關(guān)于TIMP3在奶山羊卵泡中是否表達(dá),表達(dá)規(guī)律如何,表達(dá)調(diào)控信號(hào)通路及其表達(dá)產(chǎn)物調(diào)節(jié)卵泡激素合成和顆粒細(xì)胞凋亡的分子機(jī)制等方面的研究,尚未見報(bào)道。本研究以關(guān)中奶山羊卵泡及其顆粒細(xì)胞為研究對(duì)象,在克隆和分析TIMP3基因cDNA序列的基礎(chǔ)上,分析TIMP3在卵泡周期中的表達(dá)規(guī)律,探究LH/hCG和mi RNA對(duì)TIMP3基因表達(dá)、TIMP3基因表達(dá)產(chǎn)物對(duì)卵泡激素合成、胞外基質(zhì)重構(gòu)和顆粒細(xì)胞凋亡等生物過(guò)程相關(guān)基因表達(dá)的分子調(diào)控機(jī)制,以及TIMP3基因表達(dá)產(chǎn)物對(duì)顆粒細(xì)胞凋亡的影響,闡明TIMP3基因在卵泡周期中的生物功能,主要獲得以下研究結(jié)果:1.TIMP3基因cDNA的克隆及其序列的生物信息學(xué)分析采用RACE技術(shù)從奶山羊卵巢中獲得TIMP3基因的cDNA,通過(guò)測(cè)序和序列分析發(fā)現(xiàn):奶山羊TIMP3基因cDNA序列全長(zhǎng)2208 bp,其中5'非翻譯區(qū)(5'UTR)長(zhǎng)348 bp,CDS區(qū)長(zhǎng)636 bp(編碼211氨基酸的蛋白),3'UTR長(zhǎng)1224 bp;預(yù)測(cè)的氨基酸序列與綿羊、牛、豬、人、小鼠和大鼠的序列的相似度分別為100%、99%、100%、99%、97%和96%;蛋白質(zhì)結(jié)構(gòu)預(yù)測(cè)發(fā)現(xiàn)其蛋白質(zhì)由一個(gè)N端結(jié)構(gòu)域和一個(gè)C端結(jié)構(gòu)域構(gòu)成,其中每個(gè)結(jié)構(gòu)域中包含6個(gè)保守的半胱氨酸殘基。2.TIMP3基因在奶山羊卵泡周期中的表達(dá)規(guī)律分析通過(guò)Real-time PCR分析發(fā)現(xiàn),TIMP3基因在奶山羊各組織中的表達(dá)存在差異,其中在輸卵管中的相對(duì)表達(dá)量最高;在卵泡中,TIMP3基因mRNA的豐度隨著卵泡的發(fā)育成熟逐漸升高,且在黃體中達(dá)到最高;比較分析發(fā)現(xiàn),TIMP3在多羔奶山羊(連續(xù)3胎每胎產(chǎn)羔3-4只)卵巢組織中的表達(dá)量顯著高于單羔奶山羊(連續(xù)3胎每胎產(chǎn)羔1-2只),顯示TIMP3基因與奶山羊產(chǎn)羔數(shù)可能密切相關(guān)。3.LH/hCG對(duì)TIMP3基因表達(dá)的分子調(diào)控機(jī)制分析采用hCG對(duì)培養(yǎng)的顆粒細(xì)胞進(jìn)行處理,Real-time PCR分析發(fā)現(xiàn),TIMP3基因mRNA的豐度分別在處理后4 h和24 h出現(xiàn)峰值;Western Blot分析發(fā)現(xiàn),TIMP3蛋白豐度隨著處理時(shí)間延長(zhǎng)而增加,24 h時(shí)達(dá)到最大;采用抑制劑進(jìn)行阻斷分析,發(fā)現(xiàn)hCG誘導(dǎo)的TIMP3的增加受PKA,PKC,MAPK和PI3K信號(hào)通路的影響。進(jìn)一步對(duì)TIMP3基因轉(zhuǎn)錄調(diào)控區(qū)進(jìn)行分析,發(fā)現(xiàn)TIMP3基因上游-1至-122bp的區(qū)域內(nèi),包含3個(gè)Sp1結(jié)合位點(diǎn),定點(diǎn)突變分析,發(fā)現(xiàn)-67~-58和-74~-65的突變明顯降低TIMP3的轉(zhuǎn)錄;采用cAMP激活劑FSK處理顆粒細(xì)胞,促進(jìn)Sp1表達(dá),發(fā)現(xiàn)TIMP3轉(zhuǎn)錄增強(qiáng),該結(jié)果與EMSA,ChIP以及Sp1過(guò)表達(dá)和沉默分析相一致,由此確定cAMP通過(guò)調(diào)節(jié)轉(zhuǎn)錄因子Sp1來(lái)調(diào)節(jié)TIMP3表達(dá)。4.miRNA對(duì)TIMP3基因表達(dá)的分子調(diào)控機(jī)制分析依據(jù)生物信息學(xué)分析的結(jié)果,構(gòu)建熒光素酶報(bào)告載體并與miRNAs共轉(zhuǎn)進(jìn)顆粒細(xì)胞中,發(fā)現(xiàn)miR-21,miR-221,miR-222和miR-181b均顯著的抑制了熒光素酶的活性;采用Real-time PCR分析發(fā)現(xiàn),與NC組相比miR-21和miR-181b顯著減少了顆粒細(xì)胞中TIMP3基因mRNA的豐度,而miR-221和miR-222組差異不顯著;Western Blot分析發(fā)現(xiàn),與NC組相比miR-21,miR-181b,mi R-221和miR-222都顯著減少了TIMP3蛋白的豐度,表明miR-21,miR-221,miR-222和mi R-181b與TIMP3基因3'UTR區(qū)特異位點(diǎn)結(jié)合,調(diào)控TIMP3基因的表達(dá),其中miR-21和miR-181b通過(guò)促進(jìn)mRNA降解調(diào)節(jié)TIMP3基因表達(dá),而miR-221和mi R-222通過(guò)抑制翻譯調(diào)節(jié)TIMP3的表達(dá),且miR-21,miR-221,miR-222和miR-181b可促進(jìn)顆粒細(xì)胞的活力。5.TIMP3基因表達(dá)產(chǎn)物對(duì)顆粒細(xì)胞凋亡和激素合成的分子調(diào)控機(jī)制分析采用腺病毒介導(dǎo)TIMP3基因過(guò)表達(dá)和siRNA沉默TIMP3基因表達(dá),發(fā)現(xiàn)TIMP3基因過(guò)表達(dá)可抑制顆粒細(xì)胞的活力,促進(jìn)細(xì)胞凋亡;TIMP3下調(diào)可抑制hCG誘導(dǎo)類固醇激素合成酶StAR,p450scc,HSD3B基因的表達(dá),降低孕酮水平;同時(shí)TIMP3抑制ADAM17基因表達(dá),促進(jìn)ECM蛋白相關(guān)基因FN和DCN基因的表達(dá)。以上研究表明,LH/hCG與顆粒細(xì)胞上的受體LHR結(jié)合,激活cAMP信號(hào)通路,促進(jìn)轉(zhuǎn)錄因子Sp1表達(dá),Sp1與TIMP3基因Sp1應(yīng)答元件結(jié)合,促進(jìn)TIMP3基因表達(dá),TIMP3蛋白通過(guò)促進(jìn)StAR,p450scc,HSD3B基因的表達(dá)調(diào)節(jié)孕酮的合成,促進(jìn)FN和DCN基因表達(dá)調(diào)節(jié)ECM的重構(gòu),抑制ADAM17基因表達(dá),抑制顆粒細(xì)胞的活力,促進(jìn)顆粒細(xì)胞凋亡參與卵泡周期的調(diào)控。這些研究結(jié)果,為進(jìn)一步闡明TIMP3參與調(diào)控卵泡周期的分子機(jī)理,揭示卵泡發(fā)育成熟和排卵的分子調(diào)控機(jī)制提供理論和試驗(yàn)依據(jù)。
[Abstract]:Matrix metalloproteinases (MMPs) play an important role in folliculogenesis, development, ovulation and luteal formation. As an inhibitor of MMPs, TIMP3 may participate in the coordination of various life activities during the follicular cycle. However, whether TIMP3 is expressed in the follicles of dairy goats, how it is expressed, the signaling pathway and its regulation are also involved. The expression products regulate the synthesis of follicular hormones and the molecular mechanism of apoptosis of granulosa cells. In this study, the follicles and granulosa cells of Guanzhong Dairy Goat were selected as the research object. On the basis of cloning and analyzing the cDNA sequence of TIMP3 gene, the expression of TIMP3 in follicular cycle was analyzed, and the effects of LH/hCG and MIRNA on TIMP were explored. 3 gene expression, TIMP3 gene expression products on follicular hormone synthesis, extracellular matrix remodeling and granulosa cell apoptosis and other biological processes related gene expression of molecular regulatory mechanisms, as well as TIMP3 gene expression products on granulosa cell apoptosis, to clarify the biological function of TIMP3 gene in the follicular cycle, the main results are as follows: 1. The cloning and bioinformatics analysis of the MP3 gene cDNA were carried out by RACE. The TIMP3 gene cDNA was obtained from the ovary of dairy goats. By sequencing and sequence analysis, it was found that the total length of the TIMP3 gene was 2208 bp, of which the 5'untranslated region (5'UTR) was 348 bp, the CDS region was 636 BP (encoded 211 amino acid protein), and the 3'UTR was 1224 bp. The similarity of amino acid sequences with sheep, cattle, pigs, humans, mice and rats was 100%, 99%, 100%, 99%, 97% and 96% respectively. Protein structure prediction revealed that the protein was composed of a N terminal domain and a C terminal domain, each containing 6 conserved cysteine residues.2.TIMP3 gene in dairy goat follicles. The expression pattern of TIMP3 gene in different tissues of dairy goats was analyzed by Real-time PCR, and the relative expression of TIMP3 gene was the highest in oviduct, and the abundance of TIMP3 gene mRNA in follicle increased gradually with the development and maturation of follicle, and reached the highest level in corpus luteum. The expression of TIMP3 gene in ovaries of multiple lambs dairy goats (3-4 lambs per litter) was significantly higher than that of single lamb dairy goats (1-2 lambs per litter of three consecutive fetuses). It was suggested that TIMP3 gene might be closely related to litter size of dairy goats. 3. The molecular regulation mechanism of TIMP3 gene expression by LH / hCG was analyzed by hCG in cultured granulosa cells, Real-time P CR analysis showed that the mRNA abundance of TIMP3 gene peaked at 4 h and 24 h after treatment respectively; Western Blot analysis showed that the protein abundance of TIMP3 increased with the prolongation of treatment time and reached the maximum at 24 h; blocking analysis showed that the increase of TIMP3 induced by hCG was affected by PKA, PKC, MAPK and PI3K signaling pathways. The transcriptional regulatory region of IMP3 gene was analyzed. Three Sp1 binding sites were found in the region from - 1 BP to - 122 bp upstream of TIMP3 gene. Site-directed mutagenesis analysis showed that mutations of - 67 ~ - 58 and - 74 ~ - 65 significantly reduced the transcription of TIMP3. The granulosa cells were treated with cAMP activator FSK to promote the expression of Sp1, and the transcription of TIMP3 was enhanced with EMSA, ChIP and Sp. The results of bioinformatics analysis showed that the luciferase reporter vector was constructed and co-transfected with microRNAs into granulosa cells. It was found that the expression of TIMP3 was regulated by cAMP through regulating the transcription factor Sp1. Luciferase activity was significantly inhibited; Real-time PCR analysis showed that compared with NC group, microarray-21 and microarray-181b significantly reduced the abundance of TIMP3 gene mRNA in granulosa cells, while microarray-221 and microarray-222 groups showed no significant difference; Western Blot analysis showed that compared with NC group, microarray-21, microarray-181b, microarray-221 and microarray-222 significantly reduced TIMP3 protein. Mi-21, Mi-221, Mi-222 and Mi-R-181b bind to specific sites in the 3'UTR region of the TIMP3 gene to regulate the expression of TIMP3 gene. Mi-21 and Mi-181b regulate the expression of TIMP3 gene by promoting the degradation of mRNA, while Mi-221 and Mi-R-222 regulate the expression of TIMP3 by inhibiting translation, and Mi-21, Mi-221, Mi-222 and Mi-181b promote granules. Cell viability. 5. Molecular regulatory mechanisms of TIMP3 gene expression products on apoptosis and hormone synthesis in granulosa cells were analyzed. Adenovirus-mediated TIMP3 gene overexpression and siRNA silencing of TIMP3 gene expression were used. Overexpression of TIMP3 gene inhibited the viability of granulosa cells and promoted cell apoptosis; down-regulation of TIMP3 gene inhibited hCG-induced steroid hormone expression. Synthase StAR, p450scc, HSD3B gene expression and progesterone level were decreased, while TIMP3 inhibited the expression of ADAM17 gene and promoted the expression of FN and DCN genes related to ECM protein. These studies showed that LH/hCG binds to receptor LHR on granulosa cells, activates cAMP signaling pathway, promotes the expression of transcription factor Sp1, and Sp1 response element junction between Sp1 and TIMP3 gene. TIMP3 protein regulates progesterone synthesis by promoting the expression of StAR, P450scc and HSD3B genes, promotes the expression of FN and DCN genes to regulate ECM reconstruction, inhibits the expression of ADAM17 gene, inhibits the activity of granulosa cells, and promotes the apoptosis of granulosa cells to participate in the regulation of follicular cycle. The molecular mechanism involved in the regulation of follicular cycle provides theoretical and experimental basis for revealing the molecular mechanism of follicular development and maturation and ovulation.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S827
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本文編號(hào)：2196765