热应激及神经肽Kisspeptin干预的代谢组学研究

发布时间：2018-08-09 18:49

【摘要】：应激通过高度活化神经内分泌免疫网络,扰乱内环境代谢平衡。热应激作为其中的一种,在人类方面,热应激主要集中在高温天气,会引起中暑,呼吸急促,心力衰竭,炎症等等,具有很高的致死率。在畜牧业,高热环境使动物摄食量降低,免疫力和生产力下降,造成家畜生长性能和繁殖性能的降低,会造成巨大的经济损失。Kisspeptin是由Kiss-1基因编码的一种神经肽类物质,研究发现其不仅在抑制肿瘤转移及调控生殖方面发挥了重要作用,而且在抗氧化应激中也扮演着重要角色。由于热应激是一种系统性的全身反应,涉及到整个神经内分泌免疫网络,影响多个代谢途径,单一依靠相关理化指标的测定,往往不能反映机体整体上的变化。鉴于此,本实验基于GC/TOF-MS技术的代谢组学研究方法对神经肽Kisspeptin干预大鼠热应激的模型进行了较为系统的研究。实验一:热应激对大鼠系统性及生殖相关内源性代谢物的影响。热应激是机体整个系统对外界刺激产生的全身性应答。本研究试图建立热应激的代谢组学评估方法,基于气相色谱-飞行时间质谱联用技术(GC/TOFMS)代谢技术测定热应激后SD大鼠的血清、下丘脑和附睾三种样本,得到小分子代谢物的图谱,结合多元和单元统计分析,筛选出差异性小分子代谢物。结果发现其与能量代谢、氨基酸神经递质和单胺神经递质途径密切相关,并且相关代谢物的变化与交感肾上腺素能神经内分泌系统,下丘脑-垂体-肾上腺轴及下丘脑-垂体-睾丸轴的变化相一致。同时附睾内相关代谢物的上调,可能反映出生殖系统通过代偿性保护机制的调节来纠正热应激引起的损伤。实验二:Kisspeptin对热应激大鼠肝脏氧化损伤的缓解作用。本章基于代谢组学技术结合多种理化指标和形态学的方法考察了热应激对大鼠肝组织内源性代谢物的影响。在理化指标方面,热应激组与对照组相比,GSH-PX、T-SOD水平降低(p0.05),而注射Kisspeptin后显著升高(p0.05),MDA的变化则相反;进一步从形态学观察,热应激后大鼠肝脏HE观察发现肝窦增宽增厚,周围血管充血,肝细胞开始出现水泡样变性和坏死,而Kisspeptin组和对照组相比无明显差异;透射电镜观察肝细胞的超微结构,发现热应激后,胞质中内质网排列紊乱并出现断裂,数量减少,线粒体内膜消失,并伴有空白区和胞膜增厚等表现;代谢组学结果表明热应激组与对照组在PCA图上有明显的分离趋势,而Kisspeptin组呈现回归对照组的趋势。通过采用多维统计和单维统计结合的方法,挖掘出了与热应激相关的小分子代谢物,并发现这些代谢物与脂肪酸代谢、嘌呤分解代谢及能量代谢密切相关,尤其是集中在在花生四烯酸(Arachidonate)、5,8,11,14-二十碳四烯酸(5,8,11,14-Eicosatetraenoate)、次黄嘌呤核苷(Inosine)、次黄嘌呤(Hypoxanthine)、尿素囊(Allantoin)等代谢物上,并且这些代谢物的变化在注射Kisspeptin后,与热应激组相比显著降低。本小结从代谢组学的角度研究了热应激对肝组织代谢物谱的变化,同时证实了 Kisspeptin对热应激的缓解作用。
[Abstract]:Stress is used to activate the neuroendocrine immune network and disrupt the metabolic balance of the internal environment. Heat stress is one of them. In humans, heat stress is mainly concentrated in high temperature weather, which can cause heat stroke, shortness of breath, heart failure, inflammation and so on. It has a high mortality rate. In animal husbandry, the high heat environment reduces the food intake and exempts the animals. The decline of Phytophthora and productivity, resulting in a decrease in the growth performance and reproductive performance of domestic animals, resulting in huge economic loss,.Kisspeptin is a neuropeptide encoded by the Kiss-1 gene. The study found that it not only plays an important role in inhibiting tumor metastasis and regulating reproduction, but also plays an important role in antioxidant stress. Role. Because heat stress is a systemic systemic reaction, involving the whole neuroendocrine immune network, affecting multiple metabolic pathways, and relying solely on the determination of related physical and chemical indexes, it often fails to reflect the whole changes of the body. In view of this, this experiment is based on the metabolomics research method of GC/TOF-MS technique to the neuropeptide Kisspeptin. The effect of heat stress on systemic and reproductive related endogenous metabolites in rats. Heat stress is the systemic response to external stimuli by the whole system. This study attempts to establish a metabonomics evaluation method for heat stress, based on GC flight time. Mass spectrometry (GC/TOFMS) technique was used to determine three samples of serum, hypothalamus and epididymis of SD rats after heat stress, and the spectrum of small molecule metabolites was obtained. The small molecular metabolites were screened by multivariate and unit statistical analysis. The results were found to be closely related to the pathway of energy metabolism, amino acid neurotransmitter and monoamine neurotransmitter. The changes in the related metabolites are consistent with the changes in the sympathetic adrenergic neuroendocrine system, the hypothalamus pituitary adrenal axis and the hypothalamus pituitary testicular axis. At the same time, the up-regulated metabolites in the epididymis may reflect the regulation of compensatory protection mechanism to correct the damage caused by heat stress. Experiment two: the effect of Kisspeptin on the oxidative damage of liver in rats with heat stress. In this chapter, the effects of heat stress on endogenous metabolites of liver tissues in rats were investigated by metabonomics techniques combined with various physical and chemical indexes and morphologic methods. In terms of physical and chemical indexes, the heat stress group decreased the level of GSH-PX and T-SOD (P0.05) compared with those in the group. After the injection of Kisspeptin (P0.05), the changes of MDA were the opposite. Further from the morphological observation, the liver HE observation after heat stress showed that the liver sinus was widened and thickened, the peripheral blood vessels were hyperemia, the liver cells began to appear vesicular degeneration and necrosis, while the Kisspeptin group had no significant difference compared with the control group; transmission electron microscopy observed the ultrastructure of the liver cells. After heat stress, the endoplasmic reticulum in the cytoplasm was disordered and fractured in the cytoplasm, the number of the endoplasmic reticulum was reduced, the mitochondrial intima disappeared, with the expression of the blank area and the thickening of the membrane. The metabolomics results showed that the heat stress group and the control group had a obvious separation trend on the PCA map, while the Kisspeptin group showed a tendency to return to the control group. A combination of dimensional statistics and single dimensional statistics was used to excavate small molecular metabolites associated with heat stress and found that these metabolites were closely related to fatty acid metabolism, purine catabolism and energy metabolism, especially in peanut four enoic acid (Arachidonate), 5,8,11,14- twenty carbon four enoic acid (5,8,11,14-Eicosatetraenoate), and xanthine. MTX (Inosine), hypoxanthine (Hypoxanthine), urea sac (Allantoin) and other metabolites, and the changes of these metabolites were significantly lower than those in the heat stress group after the injection of Kisspeptin. This nodule studied the changes in liver tissue metabolites from the heat stress from the metabolic point of view, and confirmed the inhibition of Kisspeptin to heat stress. Solution.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.2

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