本文選題：�；撬�　切入點(diǎn)：熱應(yīng)激　出處：《沈陽農(nóng)業(yè)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：熱應(yīng)激是造成當(dāng)前集約化養(yǎng)殖業(yè)經(jīng)濟(jì)嚴(yán)重?fù)p失的一種環(huán)境因素,對畜禽養(yǎng)殖業(yè)的長期發(fā)展影響巨大。很多動物實(shí)驗(yàn)證實(shí),熱應(yīng)激導(dǎo)致中暑死亡的很大原因在于內(nèi)皮細(xì)胞的大范圍損傷。前人的研究發(fā)現(xiàn),�；撬峋哂屑�(xì)胞保護(hù)作用,但�；撬釋τ跓釕�(yīng)激造成的內(nèi)皮細(xì)胞損傷的保護(hù)作用機(jī)制還不清楚。因此,本研究探討了�；撬釋釕�(yīng)激肉雞主動脈內(nèi)皮細(xì)胞損傷的影響。以期為治療或緩解肉雞熱應(yīng)激相關(guān)疾病等提供理論依據(jù)。試驗(yàn)選取10日齡健康肉雞,采用組織貼塊法培養(yǎng)出原代肉雞主動脈內(nèi)皮細(xì)胞,用Ⅷ因子相關(guān)抗原試劑盒經(jīng)免疫組化鑒定后,選用3-5代處在對數(shù)生長期的細(xì)胞進(jìn)行后續(xù)試驗(yàn),將內(nèi)皮細(xì)胞隨機(jī)分為五組,即正常對照(C)組、熱應(yīng)激(HS)組、熱應(yīng)激+低濃度Tau(HS+LTau)組、熱應(yīng)激+中濃度Tau(HS+MTau)組和熱應(yīng)激+高濃度Tau(HS+HTau)組,各組細(xì)胞均放入細(xì)胞培養(yǎng)箱(37℃,5%CO2)中培養(yǎng)24h,然后,正常對照組始終在37℃培養(yǎng)箱中培養(yǎng),熱應(yīng)激各組細(xì)胞同時(shí)放在43℃恒溫水浴鍋中做熱應(yīng)激處理6h,再回細(xì)胞培養(yǎng)箱中復(fù)溫培養(yǎng)1h,之后收集細(xì)胞和培養(yǎng)液上清進(jìn)行各項(xiàng)指標(biāo)的檢測。結(jié)果如下:1)�；撬釋S肉雞主動脈內(nèi)皮細(xì)胞活性和凋亡率的影響:通過MTT比色法和AO/EB染色觀察,發(fā)現(xiàn),HS組肉雞主動脈內(nèi)皮細(xì)胞活性降低、凋亡率增加。培養(yǎng)液中添加Tau的內(nèi)皮細(xì)胞相對成活率顯著高于HS組(P0.05)、內(nèi)皮細(xì)胞的凋亡率顯著低于HS組(P0.05),結(jié)合MTT比色法與AO/EB染色結(jié)果的分析,表明�；撬釋釕�(yīng)激導(dǎo)致的內(nèi)皮細(xì)胞活性降低和凋亡率增加具有明顯的抑制作用。其中,添加20mmol/L和40mmol/LTau的作用效果較好。2)�；撬釋S肉雞主動脈內(nèi)皮細(xì)胞氧化損傷的影響:與C組相比,HS組內(nèi)皮細(xì)胞LDH活性和MDA含量極顯著升高(P0.01),在抗氧化系統(tǒng)中,與C組相比,HS組內(nèi)皮細(xì)胞T-SOD活性、GSH-Px活性以及T-AOC均極顯著降低(P0.01);添加Tau后,與HS組相比,內(nèi)皮細(xì)胞的LDH活性和MDA含量均顯著降低(P0.05),內(nèi)皮細(xì)胞的T-SOD活性、GSH-Px活性和T-AOC均顯著升高(P0.05)。結(jié)果表明,HS導(dǎo)致內(nèi)皮細(xì)胞中抗氧化能力的減弱,使內(nèi)皮細(xì)胞處于嚴(yán)重的氧化損傷中,而添加Tau預(yù)防能降低內(nèi)皮細(xì)胞中LDH活性和MDA含量,并提高抗氧化物酶SOD活性、GSH-Px活性和T-AOC,抑制了內(nèi)皮細(xì)胞氧化損傷,增強(qiáng)了抗氧化能力,起到改善氧化損傷的效果。其中,添加40mmol/L Tau表現(xiàn)出較好的效果。3)�；撬釋S肉雞主動脈內(nèi)皮細(xì)胞凋亡相關(guān)基因和蛋白的影響:HS后,與C組相比,HS組內(nèi)皮細(xì)胞中Bax、Caspase-9、Caspase-3、Cyt-c和P53等促凋亡因子的基因和蛋白表達(dá)水平顯著升高(P0.05),抗凋亡因子Bcl-2基因和蛋白表達(dá)水平顯著降低(P0.05);而添加Tau后,內(nèi)皮細(xì)胞Bcl-2基因和蛋白表達(dá)水平與HS組相比顯著提高(P0.05)、促凋亡因子Bax、Caspase-9、Caspase-3、Cyt-c和P53基因和蛋白表達(dá)水平與HS組相比均顯著降低(P0.05)。綜合基因和蛋白表達(dá)水平來看,HS誘導(dǎo)了促凋亡基因和蛋白的高表達(dá),抑制了抗凋亡基因和蛋白的表達(dá),造成內(nèi)皮細(xì)胞大量凋亡,添加Tau可明顯逆轉(zhuǎn)這些變化,Tau通過提高抗凋亡因子Bcl-2的表達(dá),降低促凋亡因子Bax、Caspase-9、Caspase-3、Cyt-c和P53的表達(dá),來抑制線粒體介導(dǎo)的內(nèi)皮細(xì)胞凋亡,發(fā)揮抗凋亡作用。其中,添加40mmol/LTau對HS誘導(dǎo)的內(nèi)皮細(xì)胞凋亡的抑制作用效果較好。綜上所述,添加一定濃度的Tau能夠增強(qiáng)主動脈內(nèi)皮細(xì)胞的抗氧化能力,抑制主動脈內(nèi)皮細(xì)胞的凋亡。Tau可能通過抑制HS造成的主動脈內(nèi)皮細(xì)胞抗氧化能力的降低和線粒體介導(dǎo)的凋亡通路的活化,發(fā)揮保護(hù)主動脈內(nèi)皮細(xì)胞損傷的作用。整體比較來看,添加終濃度為40mmol/L的Tau的作用效果較好。
[Abstract]:Thermal stress is caused by an environmental factor in the current intensive breeding industry serious economic losses, the long-term development of the livestock industry a huge impact. Many animal experiments confirmed that the heat stress, resulting in a wide range of endothelial cell injury in the great cause of heatstroke death previous research found that taurine has cytoprotective effects, but the mechanism of protective effect of endothelial cell damage caused by taurine for heat stress is not clear. Therefore, this study investigated the effects of taurine on aorta endothelium in Heat Stressed Broilers. Treatment or alleviation of broiler heat stress related diseases in order to provide a theoretical basis for the test. The 10 day old healthy broilers, using tissue explant cultured method chicken aortic endothelial cells with factor VIII related antigen kit was identified by immunohistochemistry after the 3-5 generation in the logarithmic growth phase cells for subsequent Test, the endothelial cells were randomly divided into five groups: normal control group (C), heat stress (HS) group, heat stress and low concentration of Tau (HS+LTau) group, the concentration of Tau (HS+MTau) + heat stress group and the heat stress and high concentration of Tau (HS+HTau) group, each group of cells into cells incubator (at 37 5%CO2) in cultured 24h, then normal control group has 37 degrees in the incubator, the cells were placed in heat stress and constant temperature of 43 DEG C water bath heat stress 6h, then back to the cell culture box rewarming culture 1H detection after collecting cells and supernatant were the indexes. The results are as follows: 1) effect of taurine on HS activity and apoptosis rate of broiler aortic endothelial cells by MTT assay and AO/EB staining showed that HS group decreased broiler aortic endothelial cells, the apoptosis rate increased. The medium adding Tau endothelial cell relative survival rate was significantly higher than group HS (P0.05 ),鍐呯毊緇嗚優(yōu)鐨勫噵浜＄巼鏄捐憲浣庝簬HS緇，

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