绵羊甲状腺中褪黑素的合成及其生理作用的研究

发布时间：2018-05-16 02:26

本文选题：绵羊 + 甲状腺　；参考：《石河子大学》2015年硕士论文

【摘要】：褪黑素(MEL)和甲状腺素(TH)是绵羊季节性繁殖调控通路中的两个重要激素。MEL主要由松果体合成并分泌,在其过程中,5-羟色胺-N-乙酰基转移酶(AA-NAT)和羟基吲哚-氧-甲基转移酶(HIOMT)是其最关键的两个酶。AA-NAT是合成过程中的限速酶,而HIOMT是全部过程中的关键酶。TH则来自于甲状腺的合成与分泌,并受促甲状腺素释放激素(TRH),促甲状腺素(TSH)的调控。最近在实验动物小鼠的研究中证实,甲状腺存在有合成MEL所必须的AA-NAT和HIOMT两个关键酶,并分布有褪黑素受体(MT1)。同时,在人和小鼠的甲状腺存在TRH及受体(TRHR),以及促甲状腺素β亚基(TSH-β)的剪接变体,暗示甲状腺本身可能存在对TH分泌自主调节的机制以及甲状腺合成的MEL可能参与了TH的合成调节。为了研究绵羊甲状腺能否通过MEL自主调控TH的合成,进而影响绵羊季节性繁殖,开展如下研究:1、利用RT-PCR技术检测MEL合成的关键酶基因AA-NAT和HIOMT,以及MT1和调控TH合成的相关基因TRH,TRHR,TSH-β,TSHR是否在绵羊甲状腺表达。在m RNA水平确定绵羊甲状腺能否自主合成且分泌MEL,并参与调控TH合成。结果表明:绵羊甲状腺表达MEL合成的关键酶基因和MEL受体基因,以及调控TH合成的基因TRH,TRHR和TSHR,但不表达TSH-β基因。2、通过双酶常规消化并分离得到甲状腺滤泡细胞,结合RT-PCR、细胞免疫荧光和ELISA技术,对培养的绵羊甲状腺细胞从形态,甲状腺特异性基因m RNA和蛋白的表达以及甲状腺素T3/T4的合成与分泌功能等方面进行鉴定,确定所培养的原代细胞功能完整性,建立绵羊甲状腺原代细胞培养体系。结果表明:成功地建立了简便可行的绵羊甲状腺细胞的原代培养方法。为深入研究绵羊甲状腺的功能提供了实验平台。3、采用组织免疫荧光,Western blot,ELISA等技术,验证绵羊甲状腺组织中MEL合成的关键酶基因AA-NAT、HIOMT以及MT1的表达,进而利用体外培养的细胞,在细胞水平验证绵羊甲状腺能否自主合成并分泌MEL,为进一步研究绵羊甲状腺自身合成并分泌的MEL与TH合成的关系打下基础。结果表明:绵羊甲状腺具有自主合成并分泌MEL的功能,绵羊甲状腺内部可能存在有通过MEL调控TH合成的新通路。4、运用组织免疫荧光,ELISA,q PCR技术,检测外培养的绵羊甲状腺原代细胞中TSH-β蛋白的表达,并用外源MEL对体外培养的绵羊甲状腺原代细胞进行处理,检测细胞培养基中T3/T4的水平和甲状腺细胞TPO的表达量。确定绵羊甲状腺自主合成并分泌的MEL是否调控TH的合成。结果表明:绵羊甲状腺组织不表达TSH-β蛋白,自主合成并分泌的MEL在体外细胞水平与TH的合成之间没有关联,但是在外源MEL的刺激下,TPO的相对表达量与对照组差异显著,表明MEL可能参与到甲状腺滤泡细胞的抗氧化过程。综上所述,我们的研究表明,绵羊甲状腺能够自主合成并分泌MEL,但绵羊甲状腺只部分表达通过MEL通路调控TH合成的相关基因,因此,甲状腺中合成的MEL可能不直接参与调控TH合成。但在外源MEL的刺激下,TPO的表达量增加,表明甲状腺自身合成并分泌的MEL可能参与到甲状腺细胞的抗氧化作用。甲状腺中合成的MEL是否参与绵羊甲状腺其它自分泌或者旁分泌调控,甚至对参与绵羊季节性繁殖调控还有待进一步验证。
[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.

【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S826

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