本文選題：綿羊 + 甲狀腺　； 參考：《石河子大學(xué)》2015年碩士論文

【摘要】：褪黑素(MEL)和甲狀腺素(TH)是綿羊季節(jié)性繁殖調(diào)控通路中的兩個(gè)重要激素。MEL主要由松果體合成并分泌,在其過(guò)程中,5-羥色胺-N-乙酰基轉(zhuǎn)移酶(AA-NAT)和羥基吲哚-氧-甲基轉(zhuǎn)移酶(HIOMT)是其最關(guān)鍵的兩個(gè)酶。AA-NAT是合成過(guò)程中的限速酶,而HIOMT是全部過(guò)程中的關(guān)鍵酶。TH則來(lái)自于甲狀腺的合成與分泌,并受促甲狀腺素釋放激素(TRH),促甲狀腺素(TSH)的調(diào)控。最近在實(shí)驗(yàn)動(dòng)物小鼠的研究中證實(shí),甲狀腺存在有合成MEL所必須的AA-NAT和HIOMT兩個(gè)關(guān)鍵酶,并分布有褪黑素受體(MT1)。同時(shí),在人和小鼠的甲狀腺存在TRH及受體(TRHR),以及促甲狀腺素β亞基(TSH-β)的剪接變體,暗示甲狀腺本身可能存在對(duì)TH分泌自主調(diào)節(jié)的機(jī)制以及甲狀腺合成的MEL可能參與了TH的合成調(diào)節(jié)。為了研究綿羊甲狀腺能否通過(guò)MEL自主調(diào)控TH的合成,進(jìn)而影響綿羊季節(jié)性繁殖,開(kāi)展如下研究:1、利用RT-PCR技術(shù)檢測(cè)MEL合成的關(guān)鍵酶基因AA-NAT和HIOMT,以及MT1和調(diào)控TH合成的相關(guān)基因TRH,TRHR,TSH-β,TSHR是否在綿羊甲狀腺表達(dá)。在m RNA水平確定綿羊甲狀腺能否自主合成且分泌MEL,并參與調(diào)控TH合成。結(jié)果表明:綿羊甲狀腺表達(dá)MEL合成的關(guān)鍵酶基因和MEL受體基因,以及調(diào)控TH合成的基因TRH,TRHR和TSHR,但不表達(dá)TSH-β基因。2、通過(guò)雙酶常規(guī)消化并分離得到甲狀腺濾泡細(xì)胞,結(jié)合RT-PCR、細(xì)胞免疫熒光和ELISA技術(shù),對(duì)培養(yǎng)的綿羊甲狀腺細(xì)胞從形態(tài),甲狀腺特異性基因m RNA和蛋白的表達(dá)以及甲狀腺素T3/T4的合成與分泌功能等方面進(jìn)行鑒定,確定所培養(yǎng)的原代細(xì)胞功能完整性,建立綿羊甲狀腺原代細(xì)胞培養(yǎng)體系。結(jié)果表明:成功地建立了簡(jiǎn)便可行的綿羊甲狀腺細(xì)胞的原代培養(yǎng)方法。為深入研究綿羊甲狀腺的功能提供了實(shí)驗(yàn)平臺(tái)。3、采用組織免疫熒光,Western blot,ELISA等技術(shù),驗(yàn)證綿羊甲狀腺組織中MEL合成的關(guān)鍵酶基因AA-NAT、HIOMT以及MT1的表達(dá),進(jìn)而利用體外培養(yǎng)的細(xì)胞,在細(xì)胞水平驗(yàn)證綿羊甲狀腺能否自主合成并分泌MEL,為進(jìn)一步研究綿羊甲狀腺自身合成并分泌的MEL與TH合成的關(guān)系打下基礎(chǔ)。結(jié)果表明:綿羊甲狀腺具有自主合成并分泌MEL的功能,綿羊甲狀腺內(nèi)部可能存在有通過(guò)MEL調(diào)控TH合成的新通路。4、運(yùn)用組織免疫熒光,ELISA,q PCR技術(shù),檢測(cè)外培養(yǎng)的綿羊甲狀腺原代細(xì)胞中TSH-β蛋白的表達(dá),并用外源MEL對(duì)體外培養(yǎng)的綿羊甲狀腺原代細(xì)胞進(jìn)行處理,檢測(cè)細(xì)胞培養(yǎng)基中T3/T4的水平和甲狀腺細(xì)胞TPO的表達(dá)量。確定綿羊甲狀腺自主合成并分泌的MEL是否調(diào)控TH的合成。結(jié)果表明:綿羊甲狀腺組織不表達(dá)TSH-β蛋白,自主合成并分泌的MEL在體外細(xì)胞水平與TH的合成之間沒(méi)有關(guān)聯(lián),但是在外源MEL的刺激下,TPO的相對(duì)表達(dá)量與對(duì)照組差異顯著,表明MEL可能參與到甲狀腺濾泡細(xì)胞的抗氧化過(guò)程。綜上所述,我們的研究表明,綿羊甲狀腺能夠自主合成并分泌MEL,但綿羊甲狀腺只部分表達(dá)通過(guò)MEL通路調(diào)控TH合成的相關(guān)基因,因此,甲狀腺中合成的MEL可能不直接參與調(diào)控TH合成。但在外源MEL的刺激下,TPO的表達(dá)量增加,表明甲狀腺自身合成并分泌的MEL可能參與到甲狀腺細(xì)胞的抗氧化作用。甲狀腺中合成的MEL是否參與綿羊甲狀腺其它自分泌或者旁分泌調(diào)控,甚至對(duì)參與綿羊季節(jié)性繁殖調(diào)控還有待進(jìn)一步驗(yàn)證。
[Abstract]:Melatonin (MEL) and thyroxine (TH) are two important hormones in the regulation pathway of seasonal reproduction in sheep..MEL is synthesized and secreted mainly by the pineal body. In the process, 5- HT -N- acetyltransferase (AA-NAT) and hydroxyl indole oxygen methyltransferase (HIOMT) are the most critical two enzymes,.AA-NAT, the speed limiting enzyme in the synthesis process, and HI OMT is the key enzyme in the whole process,.TH is derived from the synthesis and secretion of thyroid gland, and is regulated by thyrotropin releasing hormone (TRH) and thyrotropin (TSH). Recently, in experimental animal mice, two key enzymes of AA-NAT and HIOMT, which were necessary for the synthesis of MEL, were found in the experimental animal mice and distributed in the presence of melatonin receptor (MT1). In the thyroid gland of human and mouse, TRH and receptor (TRHR), and thyroid stimulating hormone beta subunit (TSH- beta) splicing variants suggest that thyroid itself may exist autonomic regulation of TH secretion and thyroid synthesis MEL may be involved in the synthesis of TH. In order to study the independent regulation of TH synthesis by MEL in sheep's thyroid gland, The following studies were carried out in order to affect the seasonal reproduction of sheep. 1, the key enzyme genes of MEL synthesis were detected by RT-PCR technology, AA-NAT and HIOMT, as well as TRH, TRHR, TSH- beta and TSHR in the thyroid gland, which were related to MT1 and the regulation of TH synthesis, and whether the thyroid gland could be synthesized and secreted at the m RNA level. The results showed that the thyroid gland expressed the key enzyme genes of MEL synthesis and the MEL receptor gene, as well as the gene TRH, TRHR and TSHR that regulate the synthesis of TH, but did not express the TSH- beta gene.2. The thyroid follicle cells were digested and separated by double enzymes, combined with RT-PCR, cell immunofluorescence and ELISA technology. Morphology, the expression of thyroid specific gene m RNA and protein, and the synthesis and secretion of thyroxine T3/T4 were identified, the functional integrity of the cultured primary cells was determined, and the primary culture system of the sheep thyroid gland was established. The results showed that the primary culture of simple and feasible sheep thyroid cells was successfully established. Methods. In order to further study the function of the sheep thyroid gland, the experimental platform.3 was provided, using tissue immunofluorescence, Western blot, ELISA and other techniques to verify the expression of the key enzyme genes of MEL synthesis in the thyroid tissue of sheep, AA-NAT, HIOMT and MT1, and then use the cultured cells in vitro to verify the self synthesis of the sheep thyroid at the cell level. And secreting MEL to further study the relationship between MEL and TH synthesis in the synthesis and secretion of the sheep thyroid gland. The results show that the sheep thyroid gland has the function of self synthesis and secretion of MEL. There may be a new pathway.4 in the thyroid gland of the sheep's thyroid gland, which regulates the synthesis of TH by MEL, and uses tissue immunofluorescence, ELISA, Q PCR technology and detection. The expression of TSH- beta protein in the original goiter cells of sheep was cultured, and the exogenous MEL was used to treat the primary cultured thyroid cells in vitro. The level of T3/T4 in the cell culture medium and the expression of TPO in the thyroid cells were detected. The synthesis and secretion of MEL from the thyroid gland of sheep was determined to regulate the synthesis of TH. The thyroid tissue of sheep does not express TSH- beta protein, and there is no association between the level of MEL in vitro and the synthesis of TH in vitro, but the relative expression of TPO is significantly different from that of the control group under the stimulation of exogenous MEL, indicating that MEL may be involved in the antioxidant process of thyroid follicle cells. The sheep thyroid gland can synthesize and secrete MEL autonomously, but the sheep thyroid gland only partially expresses the related genes that regulate the synthesis of TH through the MEL pathway. Therefore, the MEL synthesized in the thyroid gland may not be directly involved in the regulation of TH synthesis. But under the stimulation of the exogenous MEL, the expression of TPO is increased, and the MEL that is synthesized and secreted by the thyroid gland may be involved. The antioxidant effect of thyroid cells. Whether the thyroid MEL is involved in other autocrine or paracrine regulation of the sheep thyroid gland, and even to participate in the regulation of seasonal reproduction in sheep needs to be further verified.

【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：S826

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