本文選題：非酯化脂肪酸 + 牛皺胃平滑肌細胞��； 參考：《吉林大學》2015年碩士論文

【摘要】：奶牛皺胃變位(displaced abomasum, DA)是臨床上常見的內(nèi)科疾病，該病通常發(fā)生于圍產(chǎn)期奶牛。近年來，隨著現(xiàn)代奶牛業(yè)工業(yè)化和集約化發(fā)展，飼養(yǎng)規(guī)模的迅速擴大，皺胃變位發(fā)病率呈現(xiàn)上升趨勢。該病已受到業(yè)內(nèi)人士的高度關注，其病因和發(fā)病機理是目前的研究熱點。目前研究認為皺胃遲緩和擴張是皺胃變位發(fā)生的病理學基礎。同時，流行病學調(diào)查指出皺胃變位的奶牛通常伴有血液中高濃度的非酯化脂肪酸(non-esterified fatty acid, NEFA)和β-羥丁酸，提示NEFA和β-羥丁酸與皺胃變位的發(fā)生有相關性。 本研究以原代培養(yǎng)的牛皺胃平滑肌細胞（bovine abomasum smooth musclecell, BSMC）為研究對象，觀察生理濃度和病理濃度下的NEFA對BSMC細胞活力、氧化應激、細胞凋亡等方面的影響，并進一步探究潛在的機制。MTT實驗結(jié)果顯示，0.1~0.9mmol/L的NEFA對BSMC細胞活力無負面影響，而1.2-3.3mmol/L的NEFA處理后，細胞活力顯著下降。三個處理時間點（12小時、24小時、48小時）1.2-3.3mmol/L的NEFA都能使細胞活力明顯減少。接下來，我們進行了細胞凋亡分析，發(fā)現(xiàn)0.6mmol/L的NEFA對細胞凋亡率無顯著影響，而1.2mmol/L和1.8mmol/L的NEFA處理后，細胞凋亡率顯著增高，進一步確認了NEFA對BSMC的細胞毒性。 為了探索NEFA引起的BSMC細胞死亡是否通過氧化應激途徑，我們檢測了若干個細胞內(nèi)氧化指標和抗氧化指標，包括使用DCFH-DA熒光探針檢測胞內(nèi)活性氧水平，生物化學試劑盒檢測抗氧化酶（超氧化物歧化酶和過氧化氫酶）的活力，以及丙二醛、谷胱甘肽的含量。實驗結(jié)果顯示1.2mmol/L和1.8mmol/L的NEFA有效地降低了細胞抗氧化能力而促進了氧化因子的增高，說明NEFA引起B(yǎng)SMC細胞氧化應激。 而后，我們研究了NEFA引起B(yǎng)SMC細胞凋亡的機制。通常認為，氧化應激介導的細胞凋亡是通過激活線粒體凋亡途徑。因此，我們檢測了線粒體凋亡通路的相關因子：線粒體膜電位的變化（Rhodamine123探針），細胞色素C的分布（Western blot），凋亡蛋白caspase-9、caspase-3、PARP的激活（Western blot），促/抑凋亡蛋白Bax/Bcl-2的表達（Western blot），以及凋亡蛋白AIF的入核（免疫熒光法）。實驗結(jié)果顯示1.2mmol/L和1.8mmol/L的NEFA顯著地引起了線粒體膜電位的下降，細胞色素C的釋放，caspase-9、caspase-3、PARP的激活，Bax表達增加，Bcl-2表達減少，AIF入核，說明NEFA激活了線粒體凋亡途徑。 綜合上述實驗結(jié)果，可以確認1.2mmol/L和1.8mmol/L的NEFA顯著地引起了細胞活力的降低和細胞凋亡率的增高，胞內(nèi)抗氧化能力的下降和活性氧、丙二醛含量的升高，以及凋亡蛋白的激活。此外，，在添加抗氧化劑NAC預處理后，NEFA引起的上述事件均得到了有效地抑制，進一步確認了NEFA是通過氧化應激介導的細胞凋亡機制。本研究探討了NEFA對皺胃平滑肌細胞氧化應激的影響，為加深對NEFA生理作用的認識提供理論依據(jù)，同時為闡明DA的發(fā)病機理和防治提供新的思路。
[Abstract]:Abomasum (DAA) is a common clinical medical disease, which usually occurs in perinatal cows. In recent years, with the industrialization and intensive development of modern dairy industry and the rapid expansion of feeding scale, the incidence of abomasum displacement is on the rise. The etiology and pathogenesis of the disease have attracted much attention. Current studies suggest that abomasal retardation and dilatation are the pathological basis of abomasum displacement. At the same time, epidemiological investigation showed that abomasum abomination cows were usually accompanied by high concentrations of non-esterified fatty acid (NEFAA) and 尾 -hydroxybutyric acid, suggesting that NEFA and 尾 -hydroxybutyric acid were related to the occurrence of abomastia. In this study, the primary cultured bovine abomasal smooth muscle cells (BSMC) were used to observe the effects of NEFA at physiological and pathological concentrations on the viability, oxidative stress and apoptosis of BSMC cells. The results of MTT assay showed that NEFA (0.1 ~ 0.9mmol / L) had no negative effect on the viability of BSMC cells, but the activity of BSMC cells decreased significantly after treatment with 1.2-3.3 mmol / L NEFA. Nefa (1.2-3.3 mmol / L) significantly decreased cell viability at three treatment time points (12 hours / 24 hours and 48 hours / L). Then, we analyzed the apoptosis of BSMC and found that the NEFA of 0.6 mmol / L had no significant effect on the apoptosis rate, but the apoptosis rate was significantly increased after the treatment of 1.2 mmol / L and 1.8 mmol / L NEFA, which further confirmed the cytotoxicity of NEFA to BSMC. In order to explore whether the death of BSMC cells induced by NEFA was mediated by oxidative stress, we detected several intracellular oxidation and antioxidant indexes, including the intracellular reactive oxygen species (Ros) levels detected by DCFH-DA fluorescence probe. The activity of antioxidant enzymes (superoxide dismutase and catalase) and the contents of malondialdehyde (MDA) and glutathione (GSH) were detected by biochemical kit. The results showed that the NEFA of 1.2 mmol / L and 1.8 mmol / L effectively decreased the antioxidant capacity of BSMC cells and promoted the increase of oxidation factor, which indicated that NEFA induced oxidative stress in BSMC cells. Then we studied the mechanism of NEFA induced apoptosis of BSMC cells. It is generally believed that oxidative stress mediates apoptosis through activation of mitochondrial apoptosis. therefore We detected the related factors of mitochondrial apoptosis pathway: mitochondrial membrane potential changes in Rhodamine123 probe, distribution of cytochrome C in Western blot1, activation of apoptotic protein caspase-9, caspase-3, activation of PARP, expression of promoting / inhibiting apoptosis protein Bax / Bcl-2, and apoptosis protein. The nucleation of AIF (immunofluorescence assay). The results showed that the NEFA of 1.2 mmol / L and 1.8 mmol / L resulted in the decrease of mitochondrial membrane potential, and the activation of cytochrome C, caspase-9, caspase-3, PARP and Bax expression increased the expression of Bcl 2 and decreased the expression of AIF into the nucleus, indicating that NEFA activated the mitochondrial apoptosis pathway. Combined with the above results, it was confirmed that the NEFA of 1.2 mmol / L and 1.8 mmol / L resulted in the decrease of cell viability, the increase of apoptosis rate, the decrease of intracellular antioxidant capacity, the increase of reactive oxygen species, the increase of malondialdehyde content, and the activation of apoptotic proteins. In addition, the above events induced by NEFA were effectively inhibited after pretreatment with NAC, which confirmed that NEFA is a mechanism of apoptosis mediated by oxidative stress. In this study, the effects of NEFA on oxidative stress in abomasum smooth muscle cells were studied, which provided a theoretical basis for further understanding the physiological role of NEFA, and provided a new idea for elucidating the pathogenesis of DA and its prevention and treatment.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S858.23

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