【摘要】：應(yīng)激通過(guò)高度活化神經(jīng)內(nèi)分泌免疫網(wǎng)絡(luò),擾亂內(nèi)環(huán)境代謝平衡。熱應(yīng)激作為其中的一種,在人類方面,熱應(yīng)激主要集中在高溫天氣,會(huì)引起中暑,呼吸急促,心力衰竭,炎癥等等,具有很高的致死率。在畜牧業(yè),高熱環(huán)境使動(dòng)物攝食量降低,免疫力和生產(chǎn)力下降,造成家畜生長(zhǎng)性能和繁殖性能的降低,會(huì)造成巨大的經(jīng)濟(jì)損失。Kisspeptin是由Kiss-1基因編碼的一種神經(jīng)肽類物質(zhì),研究發(fā)現(xiàn)其不僅在抑制腫瘤轉(zhuǎn)移及調(diào)控生殖方面發(fā)揮了重要作用,而且在抗氧化應(yīng)激中也扮演著重要角色。由于熱應(yīng)激是一種系統(tǒng)性的全身反應(yīng),涉及到整個(gè)神經(jīng)內(nèi)分泌免疫網(wǎng)絡(luò),影響多個(gè)代謝途徑,單一依靠相關(guān)理化指標(biāo)的測(cè)定,往往不能反映機(jī)體整體上的變化。鑒于此,本實(shí)驗(yàn)基于GC/TOF-MS技術(shù)的代謝組學(xué)研究方法對(duì)神經(jīng)肽Kisspeptin干預(yù)大鼠熱應(yīng)激的模型進(jìn)行了較為系統(tǒng)的研究。實(shí)驗(yàn)一:熱應(yīng)激對(duì)大鼠系統(tǒng)性及生殖相關(guān)內(nèi)源性代謝物的影響。熱應(yīng)激是機(jī)體整個(gè)系統(tǒng)對(duì)外界刺激產(chǎn)生的全身性應(yīng)答。本研究試圖建立熱應(yīng)激的代謝組學(xué)評(píng)估方法,基于氣相色譜-飛行時(shí)間質(zhì)譜聯(lián)用技術(shù)(GC/TOFMS)代謝技術(shù)測(cè)定熱應(yīng)激后SD大鼠的血清、下丘腦和附睪三種樣本,得到小分子代謝物的圖譜,結(jié)合多元和單元統(tǒng)計(jì)分析,篩選出差異性小分子代謝物。結(jié)果發(fā)現(xiàn)其與能量代謝、氨基酸神經(jīng)遞質(zhì)和單胺神經(jīng)遞質(zhì)途徑密切相關(guān),并且相關(guān)代謝物的變化與交感腎上腺素能神經(jīng)內(nèi)分泌系統(tǒng),下丘腦-垂體-腎上腺軸及下丘腦-垂體-睪丸軸的變化相一致。同時(shí)附睪內(nèi)相關(guān)代謝物的上調(diào),可能反映出生殖系統(tǒng)通過(guò)代償性保護(hù)機(jī)制的調(diào)節(jié)來(lái)糾正熱應(yīng)激引起的損傷。實(shí)驗(yàn)二:Kisspeptin對(duì)熱應(yīng)激大鼠肝臟氧化損傷的緩解作用。本章基于代謝組學(xué)技術(shù)結(jié)合多種理化指標(biāo)和形態(tài)學(xué)的方法考察了熱應(yīng)激對(duì)大鼠肝組織內(nèi)源性代謝物的影響。在理化指標(biāo)方面,熱應(yīng)激組與對(duì)照組相比,GSH-PX、T-SOD水平降低(p0.05),而注射Kisspeptin后顯著升高(p0.05),MDA的變化則相反;進(jìn)一步從形態(tài)學(xué)觀察,熱應(yīng)激后大鼠肝臟HE觀察發(fā)現(xiàn)肝竇增寬增厚,周圍血管充血,肝細(xì)胞開(kāi)始出現(xiàn)水泡樣變性和壞死,而Kisspeptin組和對(duì)照組相比無(wú)明顯差異;透射電鏡觀察肝細(xì)胞的超微結(jié)構(gòu),發(fā)現(xiàn)熱應(yīng)激后,胞質(zhì)中內(nèi)質(zhì)網(wǎng)排列紊亂并出現(xiàn)斷裂,數(shù)量減少,線粒體內(nèi)膜消失,并伴有空白區(qū)和胞膜增厚等表現(xiàn);代謝組學(xué)結(jié)果表明熱應(yīng)激組與對(duì)照組在PCA圖上有明顯的分離趨勢(shì),而Kisspeptin組呈現(xiàn)回歸對(duì)照組的趨勢(shì)。通過(guò)采用多維統(tǒng)計(jì)和單維統(tǒng)計(jì)結(jié)合的方法,挖掘出了與熱應(yīng)激相關(guān)的小分子代謝物,并發(fā)現(xiàn)這些代謝物與脂肪酸代謝、嘌呤分解代謝及能量代謝密切相關(guān),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)、次黃嘌呤核苷(Inosine)、次黃嘌呤(Hypoxanthine)、尿素囊(Allantoin)等代謝物上,并且這些代謝物的變化在注射Kisspeptin后,與熱應(yīng)激組相比顯著降低。本小結(jié)從代謝組學(xué)的角度研究了熱應(yīng)激對(duì)肝組織代謝物譜的變化,同時(shí)證實(shí)了 Kisspeptin對(duì)熱應(yīng)激的緩解作用。
[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.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.2

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