H9N2亚型禽流感病毒诱导蛋鸡输卵管组织凋亡及免疫相关基因表达的研究

发布时间：2018-01-19 07:40

本文关键词： H9N2 AIV 输卵管组织凋亡 Toll样受体细胞因子趋化因子　出处：《西北农林科技大学》2015年硕士论文　论文类型：学位论文

【摘要】：H9N2亚型禽流感病毒(Avain influenza virus,AIV)能引起产蛋鸡输卵管发生严重的组织损伤,导致产蛋量和蛋品质严重下降。蛋鸡感染H9N2亚型AIV除表现咳嗽,拉稀,精神萎靡的症状外,还可造成鸡群产蛋量下降30%-80%,对蛋鸡养殖造成巨大的经济损失。输卵管是鸡蛋形成的重要场所,也是流感病毒感染潜在的靶器官。然而,目前关于H9N2亚型禽流感病毒感染导致蛋鸡输卵管功能损伤的研究鲜有报道。为揭示蛋鸡感染AIV致输卵管功能异常的机理,了解蛋鸡输卵管应答AIV感染的免疫发生过程。本研究采用点眼滴鼻的方法用H9N2亚型AIV人工感染30周龄蛋鸡,HE染色观察病毒感染后输卵管不同部位发生的组织病变;TUNEL、caspase-3检测H9N2 AIV诱导蛋鸡输卵管凋亡发生的情况;采用Real-time RT-PCR检测输卵管免疫相关基因在不同感染时间的表达变化;免疫荧光染色观察CD3+CD4+和CD3+CD8α+淋巴细胞在输卵管局部浸润情况。试验结果如下:1.H9N2亚型AIV人工感染非免健康蛋鸡,设6只为对照组。感染后分别于第1d、3d、5 d、7 d颈部放血处死6只蛋鸡。按输卵管伞部、膨大部、峡部、子宫部和阴道部取材。Real-time RT-PCR病毒载量测定和NP蛋白染色证明病毒能在输卵管各部位复制,其中膨大部病毒载量最高。HE染色发现H9N2亚型AIV引起蛋鸡输卵管5个部位都发生病理变化。膨大部和峡部可见固有层腺体细胞退化,黏膜层变薄。子宫部黏膜层变薄,基质细胞坏死脱落。阴道部上皮细胞变性、坏死,组织间隙水肿。此外,子宫部和伞部的固有层都有明显的炎性细胞浸润。2.TUNEL染色、caspase-3酶活性检测发现H9N2亚型AIV感染引起蛋鸡输卵管组织凋亡。TUNEL染色显示H9N2亚型AIV感染蛋鸡可引起输卵管伞部、膨大部、峡部、子宫部、阴道部基质细胞发生组织凋亡,且膨大部和子宫部组织凋亡明显。H9N2亚型AIV感染蛋鸡输卵管组织中caspase-3酶活性较健康鸡输卵管升高,其中以膨大部、子宫部组织凋亡明显。3.采用Real-time RT-PCR检测输卵管天然免疫模式识别受体TLR3/7/21、MDA5;细胞因子IFN-α、IFN-β、IL-2;趋化因子CXCLi1、CXCLi2、XCL1,趋化因子受体XCR1、CCR5在不同感染时间的mRNA表达变化。TLR3/7/21、MDA5的基因在输卵管5个部位呈现不同程度的调节,其中MDA5基因表达上调程度最高。IFN-β、IL-2基因在输卵管五个部位出现了不同程度的上调,而IFN-α基因表达出现下调的趋势。CXCLi1、CXCLi2、XCL1、XCR1、CCR5在5个部位都表现出上调的趋势,且膨大部的上调趋势最明显。4.免疫荧光染色观察CD3+CD4+和CD3+CD8α+淋巴细胞在输卵管局部浸润情况。感染后CD3+CD4+和CD3+CD8α+在输卵管局部逐渐增多。在膨大部黏膜上皮基部和固有层,CD3+CD8α+淋巴细胞浸润情况比CD3+CD4+更为明显。综上所述,本研究证实H9N2 AIV感染蛋鸡后病毒能在输卵管组织存活复制,且引起输卵管发生组织凋亡和其他病理损伤。此外,H9N2 AIV能够引起蛋鸡输卵管免疫相关基因的表达以及CD3+CD4+和CD3+CD8α+淋巴细胞的浸润。为了解蛋鸡感染H9N2产生的临床症状和产蛋机能失调奠定了理论基础。
[Abstract]:H9N2 subtype avian influenza virus (Avain influenza, virus, AIV) can cause the occurrence of fallopian tube laying hens severe tissue damage, resulting in a serious decline in egg production and egg quality of laying hens infected with H9N2 subtype AIV. In addition to the performance of cough, diarrhea, depression symptoms, can also cause chickens to lay eggs decreased by 30%-80%. The huge economic loss of laying hens. The fallopian tube is an important place for the formation of the egg is also infected with influenza virus target organ potential. However, the H9N2 subtype of avian influenza virus infection in chicken oviduct function injury research is rarely reported. In order to reveal the mechanism of AIV infection of laying hens dysfunction caused by fallopian tube, understanding of laying hens fallopian tube immune response to AIV infection. The method of eye nose drop by H9N2 subtype AIV infected 30 week old laying hens, HE staining in different parts of the fallopian tube observation group after virus infection Fabric TUNEL, detection of H9N2 lesions; caspase-3 AIV induced apoptosis of chicken oviduct was detected by Real-time RT-PCR tube; Fallopian immune related gene expression in different time of infection; observation of CD3+CD4+ and CD3+CD8 + lymphocytes in the fallopian tube local infiltration by immunofluorescence staining. The results are as follows: 1.H9N2 subtype AIV infected non free healthy laying hens, 6 rats were in control group. After infection respectively in 1D, 3D, 5 d, 7 d, 6 laying hens were sacrificed by neck. According to the fallopian tube fimbria, enlargement, isthmus, uterus and vagina was taken.Real-time RT-PCR virus load test and NP protein staining confirmed the virus can tube site replication in the oviduct, which at the highest viral load.HE staining indicated that H9N2 subtype AIV caused pathological changes in all 5 parts of the fallopian tube. But the layers and glands visible isthmus cell degeneration, mucosal thickness . part of uterine mucosal thickness, stromal cell necrosis. Vaginal epithelial cell degeneration, necrosis, interstitial edema. In addition, the inherent layer part of uterine and umbrella have obvious infiltration of inflammatory cells in.2.TUNEL staining, detection of Caspase-3 enzyme activity of the H9N2 subtype of AIV infection caused by chicken oviduct apoptosis.TUNEL staining H9N2 subtype AIV infection can cause the umbrella of the Department of laying hens, oviduct magnum, isthmus, uterus, vaginal stromal cells occur apoptosis, and enlargement of uterine tissue and apoptosis of.H9N2 subtype AIV infected chicken oviduct tissue caspase-3 activity than healthy chicken oviduct increased with enlargement. The uterus apoptosis was detected by Real-time RT-PCR.3. was the fallopian tube of innate immune pattern recognition receptors TLR3/7/21, MDA5; cell factor IFN- alpha, IFN- beta, IL-2; chemokine CXCLi1, CXCLi2, XCL1, chemokine receptor XCR1, expression of.TLR3/7/21 CCR5 in different time of infection mRNA, MDA5 gene 5 site showed different degrees of regulation in the expression of MDA5 gene of oviduct, the highest degree of.IFN- beta, IL-2 gene five site appeared different degree of increase in IFN- gene expression of oviduct, and appeared the downward trend of.CXCLi1 CXCLi2, XCL1, XCR1, CCR5, in 5 sites showed a upward trend, to observe the CD3+CD4+ and CD3+CD8 alpha + lymphocytes in oviduct infiltration and enlargement of the upward trend the most obvious.4. immunofluorescence staining. After infection with CD3+CD4+ and CD3+CD8 + in oviduct gradually increased. In the enlargement of the mucosal epithelium basal lamina propria and CD3+CD8 + lymphocyte infiltration was more obvious than CD3+CD4+. In conclusion, this study showed that H9N2 AIV infection of laying hens after virus replication in the fallopian tube tissue survival, and cause the fallopian tube Tissue apoptosis and other pathological injuries occur. In addition, H9N2 AIV can induce the expression of immune related genes and infiltration of CD3+CD4+ and CD3+CD8 alpha + lymphocytes in laying hens. It lays a theoretical foundation for understanding the clinical symptoms and egg production dysfunction of laying hens infected with H9N2.

【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】：2015
【分类号】：S858.31

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