【摘要】：腎上腺是動(dòng)物機(jī)體非常重要的內(nèi)分泌器官,其髓質(zhì)合成和分泌的兒茶酚胺類激素(Catecholamines, CATs)在機(jī)體維持內(nèi)穩(wěn)態(tài)平衡過程中起著關(guān)鍵的作用。已有的研究證明,包括miR-375在內(nèi)的miRNAs在調(diào)節(jié)眾多內(nèi)分泌激素(如胰島素、雌激素)的合成和分泌過程中起著非常關(guān)鍵的作用,但到目前為止,miRNAs對(duì)于CATs合成和分泌的研究較少。作者實(shí)驗(yàn)室前期研究發(fā)現(xiàn)miR-375高表達(dá)于腎上腺,但其在腎上腺中的相關(guān)生物學(xué)功能尚不明確。因此,本研究的主要目標(biāo)是研究miR-375在腎上腺中的功能及相關(guān)機(jī)理。首先,利用實(shí)時(shí)定量PCR (real-time quantitative PCR,qPCR)檢測到miR-375高表達(dá)于大鼠的腎上腺組織,并且在大鼠胚胎腎上腺的發(fā)育過程中,miR-375表達(dá)呈現(xiàn)逐漸升高趨勢,在成年時(shí)達(dá)到最高;原位雜交(in situ hybridization, ISH)方法證明了 miR-375特異性表達(dá)于腎上腺髓質(zhì)嗜鉻細(xì)胞,皮質(zhì)細(xì)胞中未檢測到陽性信號(hào)。為了進(jìn)一步研究miR-375在大鼠腎上腺髓質(zhì)細(xì)胞中的功能,我們采用大鼠束縛應(yīng)急應(yīng)激模型(immobilization stress, IMO),檢測miR-375在應(yīng)急應(yīng)激反應(yīng)中的表達(dá)水平及其對(duì)CATs的影響。結(jié)果發(fā)現(xiàn)在不同的應(yīng)急應(yīng)激過程中,腎上腺中miR-375的表達(dá)下調(diào),而大鼠血漿中CATs的水平上升。隨后我們利用人T.合成的miR-375抑制劑(miR-375-in)和模擬物(miR-375-mi)分別抑制和超表達(dá)大鼠原代髓質(zhì)細(xì)胞和腎上腺髓質(zhì)瘤細(xì)胞系PC12細(xì)胞中的miR-375,結(jié)果證明miR-375-in能促進(jìn)細(xì)胞CATs的分泌;而miR-375-mi可以顯著抑制細(xì)胞分泌CATs。另外,miR-375-in可顯著上調(diào)髓質(zhì)細(xì)胞中酪氨酸羥化酶(tyrosine hydroxylase, TH)和多巴胺-β-羥化酶(dopamine-β-hydroxylase, DBH)的表達(dá);而miR-375-mi明顯下調(diào)細(xì)胞中TH和DBH的表達(dá)。同時(shí),本研究的結(jié)果證明miR-375不參與CATs的降解及去甲腎上腺素(norepinephrine,NE)對(duì)CATs的負(fù)反饋過程。此外,采用BrdU細(xì)胞免疫化學(xué)、TUNEL檢測及qPCR的方法均發(fā)現(xiàn)miR-375不影響PC12細(xì)胞的增殖和凋亡。以上實(shí)驗(yàn)結(jié)果說明了 miR-375在腎上腺CATs合成和分泌過程中起著非常重要的調(diào)節(jié)作用。另外,通過生物信息學(xué)方法預(yù)測miR-375的靶基因?yàn)樘禺愋缘鞍?1 (specificity protein 1,Sp1),并通過雙熒光素酶報(bào)告系統(tǒng)和蛋白免疫印跡等方法驗(yàn)證了 Sp1是miR-375在大鼠腎上腺中的直接靶基因。在PC12細(xì)胞中,通過RNAi干擾實(shí)驗(yàn)和Sp1過表達(dá)實(shí)驗(yàn)證明了 Sp1參與了 miR-375對(duì)TH和DBH調(diào)節(jié)的信號(hào)通路。在IMO應(yīng)急應(yīng)激反應(yīng)中,Sp1蛋內(nèi)表達(dá)顯著上調(diào)與miR-375的表達(dá)下調(diào)呈負(fù)相關(guān),再次驗(yàn)證了 Sp1為miR-375的靶基因并參與了大鼠的應(yīng)急應(yīng)激反應(yīng)過程。綜上所述,本論文證明了 miR-375在大鼠的腎上腺發(fā)育過程中持續(xù)表達(dá),并且特異地高表達(dá)于髓質(zhì)嗜鉻細(xì)胞。miR-375參與大鼠的IMO應(yīng)急應(yīng)激反應(yīng)。miR-375通過直接作用于靶基因Sp1調(diào)控TH和DBH的表達(dá),進(jìn)而影響大鼠CATs的合成和分泌。上述研究為了解miRNAs在CATs合成和分泌調(diào)控機(jī)制提供了新的依據(jù)。
[Abstract]:Adrenal gland is an important endocrine organ in animal. The synthesis and secretion of catecholamine hormone (Catecholamines, CATs) in medulla play a key role in maintaining homeostasis. It has been proved that miRNAs including miR-375 plays a key role in regulating the synthesis and secretion of many endocrine hormones (such as insulin estrogen). However so far little has been done on the synthesis and secretion of CATs by miRNAs. We found that miR-375 is highly expressed in adrenal gland, but its biological function in adrenal gland is unclear. Therefore, the main objective of this study is to study the function and mechanism of miR-375 in adrenal gland. Firstly, the high expression of miR-375 was detected in the adrenal tissue of rats by real-time quantitative PCR (real-time quantitative PCR), and the expression of mmiR-375 increased gradually during the development of embryonic adrenal gland, and reached the highest level in adulthood. In situ hybridization (in situ hybridization, ISH) showed that miR-375 was specifically expressed in chromaffin cells of adrenal medulla, but no positive signal was detected in cortical cells. In order to further study the function of miR-375 in adrenal medullary cells of rats, the expression of miR-375 in emergency stress response and its effect on CATs were detected by rat restraint emergency stress model (immobilization stress, IMO),). The results showed that the expression of miR-375 was down-regulated in adrenal gland and the level of CATs in plasma was increased during different emergency stress. Then we use the human T. The synthesis of miR-375 inhibitor (miR-375-in) and analogue (miR-375-mi) inhibited and overexpressed miR-375in primary rat medullary cells and adrenal medulloma cell line PC12, respectively. The results showed that miR-375-in could promote the secretion of CATs, while miR-375-mi could significantly inhibit the secretion of CATs. In addition, miR-375-in significantly up-regulated the expression of tyrosine hydroxylase (tyrosine hydroxylase, TH) and dopamine 尾 -hydroxylase (DBH) in medullary cells, while miR-375-mi significantly down-regulated the expression of th and DBH in medullary cells. At the same time, the results showed that miR-375 did not participate in the degradation of CATs and the negative feedback of norepinephrine (NE) on CATs. In addition, miR-375 did not affect the proliferation and apoptosis of PC12 cells by BrdU cell immunocytochemistry and qPCR assay. These results suggest that miR-375 plays an important role in the regulation of adrenal CATs synthesis and secretion. In addition, the target gene of miR-375 was predicted by bioinformatics because of its specific protein 1 (specificity protein 1 Sp1, and the direct target gene of miR-375 in rat adrenal gland was confirmed by double luciferase report system and Western blot. In PC12 cells, RNAi interference experiments and Sp1 overexpression experiments demonstrated that Sp1 participated in the signal pathway regulated by miR-375 on th and DBH. There was a negative correlation between the up-regulation of SP1 expression and the down-regulation of miR-375 expression in the IMO emergency stress response, which confirmed that Sp1 was the target gene of miR-375 and participated in the process of emergency stress response in rats. To sum up, this paper proves that miR-375 is expressed continuously during the development of adrenal gland in rats. MiR-375 was specifically overexpressed in medullary chromaffin cells. MiR-375 participated in the IMO emergency stress response of rats. MiR-375 regulated the expression of th and DBH by directly acting on the target gene Sp1, and then affected the synthesis and secretion of CATs in rats. These studies provide a new basis for understanding the regulatory mechanism of miRNAs in the synthesis and secretion of CATs.
【學(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q454

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