本文選題：BHBA + NF-κB通路　； 參考：《吉林大學》2017年碩士論文

【摘要】：血液中高濃度的β-羥丁酸(BHBA)是圍產(chǎn)期酮病奶牛的主要臨床特征。中性粒細胞是機體抵抗外源細菌感染的第一道防線,酮病奶牛表現(xiàn)為中性粒細胞免疫活性下降,吞噬作用、呼吸爆發(fā)作用減弱,從而導致酮病奶牛易于發(fā)生感染性疾病。因此,高濃度的BHBA可能與酮病奶牛免疫細胞免疫抵抗力下降密切相關(guān)。圍產(chǎn)期酮病奶牛干物質(zhì)攝入量減少而泌乳量增加所造成能量負平衡(NEB),可影響生長激素-胰島素樣生長因子(GH-IGF)軸內(nèi)分泌調(diào)控系統(tǒng),使胰島素樣生長因子1(IGF-1)分泌減少,干擾機體能量代謝,由此可加重酮病奶牛的先天免疫抑制。IGF-1在調(diào)控細胞代謝、增殖和分化方面起著非常重要的作用,因此,我們推測給予外源性IGF-1有可能改善酮病奶牛的免疫抑制。本實驗通過體外培養(yǎng)奶牛中性粒細胞,分別添加不同濃度的BHBA、IGF-1和NF-κB通路抑制劑、PI3K抑制劑、自噬抑制劑,來探討IGF-1調(diào)控圍產(chǎn)期酮病奶牛中性粒細胞免疫抑制的作用與機制;通過分析酮病奶牛免疫抑制的原因,探究IGF-1調(diào)控中性粒細胞免疫抑制的信號通路,并通過測定中性粒細胞活性氧產(chǎn)生、吞菌實驗和呼吸爆發(fā),來揭示IGF-1在調(diào)控中性粒細胞免疫功能的作用,為提高酮病奶牛中性粒細胞免疫抑制提供理論依據(jù)。通過體內(nèi)實驗檢測奶牛血液指標,結(jié)果顯示,與健康奶牛相比,酮病奶牛血清中GH明顯升高,而IGF-1的濃度顯著下降,說明NEB導致GH調(diào)控IGF-1通路被阻斷。通過Western blotting結(jié)果分析,表明酮病奶牛中性粒細胞發(fā)生嚴重的自噬現(xiàn)象,而自噬的過度積累會引發(fā)Ⅱ型細胞程序性死亡,這可能與細胞免疫抑制有關(guān)。體外培養(yǎng)中性粒細胞添加4.8 m M BHBA結(jié)果顯示,在作用6 h時自噬關(guān)鍵蛋白LC3和磷酸化NF-κB p65蛋白表達量最高。添加不同濃度的BHBA及NF-κB通路抑制劑PDTC,結(jié)果表明,隨著BHBA濃度的升高,磷酸化NF-κB p65的表達上調(diào),加重了自噬的積累;而添加NF-κB通路抑制劑后,減輕了自噬的積累,說明高濃度的BHBA通過調(diào)控NF-κB通路上調(diào)了自噬的積累,所以,抑制NF-κB對調(diào)控中性粒細胞自噬尤為重要。隨后在高濃度的BHBA的基礎(chǔ)上,添加不同濃度的IGF-1,在IGF-1在100 ng/m L時,顯著下調(diào)了NF-κB通路并抑制自噬的發(fā)生。IGF-1信號通路上的蛋白PI3K、AKT的測定結(jié)果表明,IGF-1可以上調(diào)PI3K和磷酸化AKT的表達,而添加PI3K抑制劑LY294002后,PI3K/AKT通路被抑制,磷酸化NF-κB p65和自噬關(guān)鍵蛋白LC3顯著升高,說明IGF-1是通過調(diào)控PI3K/AKT通路和緩解NF-κB通路來抑制自噬的。通過體外添加高濃度的BHBA,檢測其對中性粒細胞的免疫活性的影響,結(jié)果表明高濃度的BHBA使中性粒細胞活性氧的產(chǎn)生、吞菌實驗和呼吸爆發(fā)作用顯著下調(diào),而添加自噬抑制劑3-MA后,抑制了高BHBA誘導的自噬積累,中性粒細胞活性氧的產(chǎn)生、吞菌實驗和呼吸爆發(fā)作用顯著上升,說明高BHBA誘發(fā)中性粒細胞自噬積累,導致細胞免疫活性下調(diào)。在高濃度的BHBA的基礎(chǔ)上添加IGF-1(100 ng/m L)后,細胞免疫活性顯著上調(diào),說明IGF-1可以通過下調(diào)自噬來緩解由BHBA引起的中性粒細胞免疫活性低下。通過以上實驗可以認為:高濃度的BHBA可以使中性粒細胞NF-κB通路激活,導致自噬過度積累,細胞免疫活性顯著下降;IGF-1可以通過調(diào)控PI3K/AKT通路和緩解NF-κB通路抑制自噬的發(fā)生,從而上調(diào)中性粒細胞活性氧產(chǎn)生、吞菌能力和呼吸爆發(fā)作用,恢復中性粒細胞的免疫活性,進而提高圍產(chǎn)期酮病奶牛先天免疫機能。
[Abstract]:The high concentration of beta hydroxybutyrate (BHBA) in the blood is the main clinical feature of perinatal ketosis cows. Neutrophils are the first line of defense against the external bacterial infection. The cows with ketosis show the decrease of neutrophil immune activity, phagocytosis and respiratory burst, which leads to the prone to infectious diseases of ketosis cows. Therefore, high concentration of BHBA may be closely related to the decrease of immune cell immunity in cows with ketodisease. The decrease of dry matter intake and the increase of lactating amount caused by the negative balance of energy (NEB) in perinatal ketosis cows may affect the regulation system of the growth hormone insulin-like growth factor (GH-IGF), and make insulin-like growth factor 1 (IGF-1). Decrease in secretion and interfere with the body's energy metabolism, thus can aggravate the congenital immunosuppressive.IGF-1 of cows with ketosis, which plays a very important role in regulating cell metabolism, proliferation and differentiation. Therefore, we speculate that giving exogenous IGF-1 may improve the immunosuppression of cows with ketosis. Do not add different concentrations of BHBA, IGF-1 and NF- kappa B pathway inhibitors, PI3K inhibitors, autophagy inhibitors, to explore the role and mechanism of IGF-1 regulation of neutrophilic granulocyte immunosuppression in perinatal cows. By analyzing the causes of immunosuppression in cows with ketoosis, the signal pathway of sexual granulocyte immunosuppression in IGF-1 regulation is explored, and the determination of the signal pathway in the regulation of sexual granulocyte immunity in IGF-1 regulation and regulation is determined. Neutrophil reactive oxygen species, bacteria swallowing experiment and respiratory burst to reveal the role of IGF-1 in regulating the immune function of neutrophils, and provide a theoretical basis for improving neutrophils immune suppression in cows with ketosis. The blood indexes of dairy cows were detected by in vivo experiments. The results showed that the serum levels of GH in cows of ketosis cows were significantly higher than those of healthy cows. The concentration of IGF-1 decreased significantly, indicating that NEB led to the blocking of the GH regulation IGF-1 pathway. Through the analysis of Western blotting results, the serious autophagy occurred in neutrophils of cows with ketodisease, and the excessive accumulation of autophagy could lead to programmed cell death of type II cells, which may be related to cellular immunity inhibition. Adding 4.8 m M BHBA results showed that the expression of autophagic key protein LC3 and phosphorylated NF- kappa B p65 protein was the highest at 6 h. The addition of BHBA and NF- kappa B pathway inhibitor PDTC, the results showed that the expression of phosphorylated kappa kappa was up up and increased the accumulation of autophagy with the increase of concentration. The accumulation of autophagy indicates that high concentration of BHBA increases the accumulation of autophagy by regulating the NF- kappa B pathway. Therefore, inhibition of NF- kappa B is particularly important for regulating autophagy in neutrophils. Then, on the basis of high concentration of BHBA, the addition of different concentrations of IGF-1 is added to IGF-1 at 100 ng/m L, which significantly reduces the NF- kappa B pathway and inhibits the occurrence of autophagy. The results of the protein PI3K on the F-1 signaling pathway, AKT showed that IGF-1 could up regulate the expression of PI3K and phosphorylated AKT, while the PI3K/AKT pathway was inhibited after the addition of the PI3K inhibitor LY294002, and the phosphorylated NF- kappa B p65 and autophagy key proteins were significantly increased, indicating that the inhibition of autophagy by regulating the pathway and alleviating the kappa pathway. The effect of high concentration of BHBA on the immune activity of neutrophils was detected in vitro. The results showed that high concentration of BHBA resulted in the production of reactive oxygen species in neutrophils, the inhibition of autophagy induced autophagy and the production of neutrophils after adding autophagic inhibitor 3-MA. BHBA induced autophagic accumulation of neutrophils and decreased cellular immune activity. After adding IGF-1 (100 ng/m L) on the basis of high concentration of BHBA, the cellular immune activity was significantly up-regulated, indicating that IGF-1 could alleviate neutrophils induced by BHBA by down regulation of autophagy. It is suggested that high concentration of BHBA can activate the NF- kappa B pathway of neutrophils, lead to excessive accumulation of autophagy, and significantly decrease the cellular immune activity; IGF-1 can inhibit the occurrence of autophagy by regulating the PI3K/AKT pathway and alleviating the NF- kappa B pathway, thus increasing the production of reactive oxygen species in neutrophils, the ability to swallow bacteria and the ability to swallow bacteria. The effect of respiratory burst can restore the immune activity of neutrophils and further improve the innate immunity of perinatal ketosis cows.

【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S858.23

【參考文獻】

相關(guān)期刊論文 前1條

1 ;Hsp90 inhibition results in autophagy-mediated proteasome-independent degradation of IκB kinase(IKK)[J];Cell Research;2006年11期

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本文編號：1850470