非酯化脂肪酸诱导牛皱胃平滑肌细胞死亡的机制研究

发布时间：2018-06-20 14:46

本文选题：非酯化脂肪酸 + 牛皱胃平滑肌细胞　；参考：《吉林大学》2015年硕士论文

【摘要】：奶牛皱胃变位(displaced abomasum, DA)是临床上常见的内科疾病，该病通常发生于围产期奶牛。近年来，随着现代奶牛业工业化和集约化发展，饲养规模的迅速扩大，皱胃变位发病率呈现上升趋势。该病已受到业内人士的高度关注，其病因和发病机理是目前的研究热点。目前研究认为皱胃迟缓和扩张是皱胃变位发生的病理学基础。同时，流行病学调查指出皱胃变位的奶牛通常伴有血液中高浓度的非酯化脂肪酸(non-esterified fatty acid, NEFA)和β-羟丁酸，提示NEFA和β-羟丁酸与皱胃变位的发生有相关性。本研究以原代培养的牛皱胃平滑肌细胞（bovine abomasum smooth musclecell, BSMC）为研究对象，观察生理浓度和病理浓度下的NEFA对BSMC细胞活力、氧化应激、细胞凋亡等方面的影响，并进一步探究潜在的机制。MTT实验结果显示，0.1~0.9mmol/L的NEFA对BSMC细胞活力无负面影响，而1.2-3.3mmol/L的NEFA处理后，细胞活力显著下降。三个处理时间点（12小时、24小时、48小时）1.2-3.3mmol/L的NEFA都能使细胞活力明显减少。接下来，我们进行了细胞凋亡分析，发现0.6mmol/L的NEFA对细胞凋亡率无显著影响，而1.2mmol/L和1.8mmol/L的NEFA处理后，细胞凋亡率显著增高，进一步确认了NEFA对BSMC的细胞毒性。为了探索NEFA引起的BSMC细胞死亡是否通过氧化应激途径，我们检测了若干个细胞内氧化指标和抗氧化指标，包括使用DCFH-DA荧光探针检测胞内活性氧水平，生物化学试剂盒检测抗氧化酶（超氧化物歧化酶和过氧化氢酶）的活力，以及丙二醛、谷胱甘肽的含量。实验结果显示1.2mmol/L和1.8mmol/L的NEFA有效地降低了细胞抗氧化能力而促进了氧化因子的增高，说明NEFA引起BSMC细胞氧化应激。而后，我们研究了NEFA引起BSMC细胞凋亡的机制。通常认为，氧化应激介导的细胞凋亡是通过激活线粒体凋亡途径。因此，我们检测了线粒体凋亡通路的相关因子：线粒体膜电位的变化（Rhodamine123探针），细胞色素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的表达（Western blot），以及凋亡蛋白AIF的入核（免疫荧光法）。实验结果显示1.2mmol/L和1.8mmol/L的NEFA显著地引起了线粒体膜电位的下降，细胞色素C的释放，caspase-9、caspase-3、PARP的激活，Bax表达增加，Bcl-2表达减少，AIF入核，说明NEFA激活了线粒体凋亡途径。综合上述实验结果，可以确认1.2mmol/L和1.8mmol/L的NEFA显著地引起了细胞活力的降低和细胞凋亡率的增高，胞内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，，在添加抗氧化剂NAC预处理后，NEFA引起的上述事件均得到了有效地抑制，进一步确认了NEFA是通过氧化应激介导的细胞凋亡机制。本研究探讨了NEFA对皱胃平滑肌细胞氧化应激的影响，为加深对NEFA生理作用的认识提供理论依据，同时为阐明DA的发病机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.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.23

【参考文献】