褪黑素对妊娠期猪黄体细胞孕酮分泌的调节作用及机制研究

发布时间：2018-07-14 10:20

【摘要】：褪黑素(melatonin)是一种伴随昼夜节律变化而产生的神经内分泌胺类激素,主要由哺乳动物松果体和视网膜分泌产生。研究表明,褪黑素主要通过其膜受体(Melatoninreceptor,MR)发挥生物学作用,1998年IUPHAR会议将哺乳动物的褪黑素受体命名为MT1、MT2和MT3三种亚型。褪黑素在下丘脑和垂体水平调节动物生殖活动深受研究者的关注,近年来的研究发现,卵巢也是褪黑素直接作用的靶器官,褪黑素膜受体在卵巢各种组织广泛分布,对卵泡的发育、雌激素和孕酮的分泌都具有调节作用,但是这些研究多集中在人和鼠,对家畜的研究较少,尤其是褪黑素对家畜妊娠期黄体功能调节作用的研究未见报道。本研究拟选择妊娠期母猪为研究对象,采用免疫组织化学染色、细胞间接免疫荧光、QRT-PCR以及westernblotting等方法研究褪黑素膜受体在妊娠期猪黄体组织和体外培养猪黄体细胞中的分布定位、褪黑素对猪黄体细胞孕酮分泌的调节作用及机制。研究结果如下。1.免疫组织化学染色结果表明,MT1和MT2广泛分布于妊娠期猪黄体组织,主要定位于黄体细胞。2.细胞间接免疫荧光结果表明,MT1和MT2在体外培养的猪黄体细胞中大量分布。细胞活性试验结果表明,生理浓度的褪黑素能显著上调黄体细胞活性(p0.05)。3.不同浓度梯度褪黑素处理黄体细胞24h,结果表明,1pg/mL、5pg/mL、25pg/mL、125pg/mL和625pg/mL的褪黑素都能显著促进孕酮的分泌(p0.05),且呈剂量依赖性。分别以25pg/mL和125pg/mL褪黑素处理黄体细胞0h、8h、16h、24h、32h、40h和48h后,结果表明,孕酮的分泌量与处理时间呈依赖性,且培养8h后的所有时间点孕酮的分泌量都显著高于对照组(p0.05)。分别以25pg/mL和125pg/m褪黑素处理黄体细胞16h,同时以褪黑素膜受体抑制剂处理黄体细胞,结果发现,褪黑素对黄体细胞孕酮分泌的促进作用完全被阻断。4.分别以25pg/mL和125pg/mL褪黑素处理黄体细胞16h,QRT-PCR和westernblotting方法检测褪黑素对孕酮合成过程中3种调节酶表达的影响。结果发现,褪黑素上调StAR和P450scc的表达,而对3β-HSD的表达没有影响;膜受体抑制剂能阻断褪黑素对StAR和P450scc表达的上调作用。本研究证实了褪黑素膜受体MT1和MT2广泛分布于妊娠期猪黄体组织,主要定位于黄体细胞,生理浓度的褪黑素能显著上调黄体细胞活性。研究发现了褪黑素通过其膜受体MT1和MT2促进了黄体细胞孕酮的分泌,且呈时间和剂量依赖性。进一步研究发现,褪黑素通过MT1和MT2促进黄体细胞孕酮分泌的作用可能是上调了StAR和P450scc来完成的。
[Abstract]:Melatonin (melatonin) (melatonin (melatonin) is a neuroendocrine amine hormone produced by circadian rhythm changes. It is mainly produced by the secretion of mammalian pineal gland and retina. The results show that melatonin mainly plays a biological role through its membrane receptor (Mr). In 1998, the IUPHAR named the mammalian melatonin receptor as three subtypes of MT1, MT2 and MT3. The regulation of animal reproductive activity by melatonin at the level of hypothalamus and pituitary has attracted much attention. In recent years, it has been found that the ovary is also a target organ directly affected by melatonin, and melatonin membrane receptors are widely distributed in various ovarian tissues. Both estrogen and progesterone secretion can regulate follicular development, but these studies mainly focus on human and mouse, but there are few studies on domestic animals, especially the regulation of melatonin on the luteal function of livestock during pregnancy has not been reported. The aim of this study was to study the distribution of melatonin membrane receptor (melatonin membrane receptor) in porcine luteal tissue and porcine luteal cells cultured in vitro by immunohistochemical staining, indirect immunofluorescence RT-PCR and westernblotting. Effects and mechanisms of melatonin on progesterone secretion in porcine luteal cells. The results are as follows. Immunohistochemical staining showed that MT1 and MT2 were widely distributed in porcine luteal tissue during pregnancy, mainly located in luteal cells. The results of cell indirect immunofluorescence showed that MT1 and MT2 were widely distributed in porcine luteal cells cultured in vitro. The results of cell activity test showed that melatonin significantly upregulated luteal cell activity (p0.05). Different concentrations of melatonin treated luteal cells for 24 h. The results showed that melatonin (125pg / mL) and melatonin (625pg / mL) significantly increased progesterone secretion (p0.05) in a dose-dependent manner. After treatment with melatonin at 25 PG / mL and 125 PG / mL for 40 h and 48 h, respectively, the secretion of progesterone in luteal cells was significantly higher than that in control group at all time points (p0.05). Luteal cells were treated with 25 PG / mL and 125pg/m for 16 h, respectively, and luteal cells were treated with melatonin membrane receptor inhibitor for 16 h. The results showed that the effect of melatonin on progesterone secretion in luteal cells was completely blocked. The effects of melatonin on the expression of three regulatory enzymes during progesterone synthesis were detected by 16h QRT-PCR and westernblotting in luteal cells treated with 25 PG / mL and 125 PG / mL melatonin, respectively. The results showed that melatonin up-regulated the expression of star and P450scc, but had no effect on the expression of 3 尾 -HSD, and the membrane receptor inhibitor blocked the up-regulation of the expression of star and P450scc by melatonin. This study confirmed that melatonin membrane receptors MT1 and MT2 were widely distributed in porcine luteal tissues during pregnancy, mainly located in luteal cells. Melatonin at physiological concentration could significantly up-regulate luteal cell activity. It was found that melatonin promoted progesterone secretion in luteal cells in a time-and dose-dependent manner through its membrane receptors MT1 and MT2. Further studies showed that melatonin promoted progesterone secretion in luteal cells by MT1 and MT2, which might be due to the up-regulation of star and P450scc.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S828

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