血红素加氧酶1对H9N2 AIV在鸡输卵管上皮细胞中增殖的调控作用

发布时间：2018-06-28 23:30

本文选题：H9N2 + AIV　；参考：《西北农林科技大学》2017年硕士论文

【摘要】：H9N2亚型禽流感为低致病性禽流感(Low Pathogenic Avian influenza,LPAI),其传染性较高、致死率较低,感染后可造成禽类的免疫力下降,呼吸道和生殖道系统的病变,蛋鸡表现为产蛋率显著下降,蛋的品质下降,产薄壳蛋、软壳蛋、砂皮蛋和畸形蛋,引发严重的经济损失。但目前尚未见关于H9N2亚型禽流感病毒感染如何导致鸡输卵管上皮细胞发生病变的分子机制研究的报道。血红素加氧酶1(Heme Oxygenase-1,HO-1)为机体内重要的抗氧化应激蛋白酶,在抗炎、抗氧化、抗细胞凋亡、抗病毒感染中都有重要的作用。因此,本实验分离培养了原代鸡输卵管上皮细胞(chicken oviduct epithelial cells,COEC),研究了H9N2 AIV感染对细胞中HO-1表达的影响,并初步探索HO-1调控H9N2 AIV增殖的机制,主要研究结果如下:1.H9N2 AIV在鸡输卵管上皮细胞中的增殖研究。成功分离培养了鸡输卵管上皮细胞,细胞表现为典型的上皮样形态,生长状态良好,角蛋白18鉴定呈阳性。H9N2 AIV感染第3代COEC,可观察到细胞产生明显的CPE,表现为细胞皱缩、拉长变细,部分细胞变圆,甚至裂解死亡。Western Blot可检测到宿主细胞中有病毒NS1蛋白的表达。2.H9N2 AIV感染COEC后细胞内HO-1的表达变化。H9N2 AIV感染COEC后,通过Real time PCR及Western Blot方法检测不同时间点HO-1的变化情况。结果表明,HO-1的mRNA和蛋白表达均水平显著增加。3.HO-1在H9N2 AIV的增殖过程中的作用研究。为了研究HO-1在H9N2 AIV增殖过程中的作用,我们首先用HO-1的抑制剂ZnPP处理COEC,Western Blot检测ZnPP抑制HO-1对H9N2 AIV增殖的影响。结果表明,ZnPP抑制了细胞中HO-1表达的同时,也显著抑制H9N2 AIV的增殖水平。之后,我们将能特异性干扰HO-1表达的siRNA转染COEC,同样显著降低了HO-1和H9N2 AIV的蛋白表达水平。综上所述,本研究表明H9N2 AIV感染可上调COEC中HO-1的表达,而下调HO-1表达可显著抑制H9N2 AIV在细胞中的增殖。研究结果为抗H9N2药物的研发提供重要的参考靶点,也为禽流感病毒的防治提供了新思路。
[Abstract]:H9N2 subtype avian influenza (H9N2) is a low pathogenic Avian influenzavirus (LPAI) with high infectivity and low mortality. It can cause the decrease of poultry immunity and the pathological changes of respiratory tract and reproductive system after infection. The laying rate of laying hens is significantly decreased. The quality of eggs declined, and the production of thin shell eggs, soft shell eggs, sand eggs and malformed eggs caused serious economic losses. However, there are no reports on the molecular mechanism of H9N2 subtype avian influenza virus infection in the pathogenesis of oviduct epithelial cell lesions in chickens. Heme Oxygenase-1 (Heme Oxygenase-1) is an important antioxidant stress protease, which plays an important role in anti-inflammation, anti-oxidation, anti-apoptosis and anti-virus infection. Therefore, we isolated and cultured primary chicken oviductal epithelial cells (chicken oviduct epithelial cells), studied the effect of H9N2 AIV infection on the expression of HO-1, and preliminarily explored the mechanism of HO-1 regulating H9N2 AIV proliferation. The main results are as follows: 1. The proliferation of H 9 N 2 AIV in chicken oviduct epithelial cells. Chicken oviduct epithelial cells were isolated and cultured successfully. The cells showed typical epithelial-like morphology and good growth state. The positive reaction of keratin 18 was observed in the third generation of COEC.H9N2 AIV infection. The expression of virus NS1 protein in host cells was detected by Western Blot. 2. The changes of HO-1 expression in host cells after infection with COEC. H9N2 AIV infected COEC. Real time PCR and Western blot were used to detect the changes of HO-1 at different time points. The results showed that the mRNA and protein expression of HO-1 increased significantly. 3. The role of HO-1 in the proliferation of H9N2 AIV was studied. In order to study the role of HO-1 in the proliferation of H9N2 AIV, we first detected the effect of inhibition of HO-1 on H9N2 AIV proliferation by ZnPP treatment with COEC-Western Blot. The results showed that ZnPP inhibited the expression of HO-1 and significantly inhibited the proliferation of H9N2 AIV. Subsequently, we transfected COECs with siRNA that specifically interfered with HO-1 expression, which also significantly reduced the protein expression level of HO-1 and H9N2 AIV. In conclusion, H9N2 AIV infection can up-regulate the expression of HO-1 in COEC, but down-regulate HO-1 expression can significantly inhibit the proliferation of H9N2 AIV in cells. The results provide an important reference target for the development of anti-H9N2 drugs and a new idea for the prevention and treatment of avian influenza virus.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.31

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