發(fā)布時間：2018-01-19 07:40

  本文關鍵詞： H9N2 AIV 輸卵管組織 凋亡 Toll樣受體 細胞因子 趨化因子　出處：《西北農林科技大學》2015年碩士論文　論文類型：學位論文

【摘要】：H9N2亞型禽流感病毒(Avain influenza virus,AIV)能引起產蛋雞輸卵管發(fā)生嚴重的組織損傷,導致產蛋量和蛋品質嚴重下降。蛋雞感染H9N2亞型AIV除表現(xiàn)咳嗽,拉稀,精神萎靡的癥狀外,還可造成雞群產蛋量下降30%-80%,對蛋雞養(yǎng)殖造成巨大的經濟損失。輸卵管是雞蛋形成的重要場所,也是流感病毒感染潛在的靶器官。然而,目前關于H9N2亞型禽流感病毒感染導致蛋雞輸卵管功能損傷的研究鮮有報道。為揭示蛋雞感染AIV致輸卵管功能異常的機理,了解蛋雞輸卵管應答AIV感染的免疫發(fā)生過程。本研究采用點眼滴鼻的方法用H9N2亞型AIV人工感染30周齡蛋雞,HE染色觀察病毒感染后輸卵管不同部位發(fā)生的組織病變;TUNEL、caspase-3檢測H9N2 AIV誘導蛋雞輸卵管凋亡發(fā)生的情況;采用Real-time RT-PCR檢測輸卵管免疫相關基因在不同感染時間的表達變化;免疫熒光染色觀察CD3+CD4+和CD3+CD8α+淋巴細胞在輸卵管局部浸潤情況。試驗結果如下:1.H9N2亞型AIV人工感染非免健康蛋雞,設6只為對照組。感染后分別于第1d、3d、5 d、7 d頸部放血處死6只蛋雞。按輸卵管傘部、膨大部、峽部、子宮部和陰道部取材。Real-time RT-PCR病毒載量測定和NP蛋白染色證明病毒能在輸卵管各部位復制,其中膨大部病毒載量最高。HE染色發(fā)現(xiàn)H9N2亞型AIV引起蛋雞輸卵管5個部位都發(fā)生病理變化。膨大部和峽部可見固有層腺體細胞退化,黏膜層變薄。子宮部黏膜層變薄,基質細胞壞死脫落。陰道部上皮細胞變性、壞死,組織間隙水腫。此外,子宮部和傘部的固有層都有明顯的炎性細胞浸潤。2.TUNEL染色、caspase-3酶活性檢測發(fā)現(xiàn)H9N2亞型AIV感染引起蛋雞輸卵管組織凋亡。TUNEL染色顯示H9N2亞型AIV感染蛋雞可引起輸卵管傘部、膨大部、峽部、子宮部、陰道部基質細胞發(fā)生組織凋亡,且膨大部和子宮部組織凋亡明顯。H9N2亞型AIV感染蛋雞輸卵管組織中caspase-3酶活性較健康雞輸卵管升高,其中以膨大部、子宮部組織凋亡明顯。3.采用Real-time RT-PCR檢測輸卵管天然免疫模式識別受體TLR3/7/21、MDA5;細胞因子IFN-α、IFN-β、IL-2;趨化因子CXCLi1、CXCLi2、XCL1,趨化因子受體XCR1、CCR5在不同感染時間的mRNA表達變化。TLR3/7/21、MDA5的基因在輸卵管5個部位呈現(xiàn)不同程度的調節(jié),其中MDA5基因表達上調程度最高。IFN-β、IL-2基因在輸卵管五個部位出現(xiàn)了不同程度的上調,而IFN-α基因表達出現(xiàn)下調的趨勢。CXCLi1、CXCLi2、XCL1、XCR1、CCR5在5個部位都表現(xiàn)出上調的趨勢,且膨大部的上調趨勢最明顯。4.免疫熒光染色觀察CD3+CD4+和CD3+CD8α+淋巴細胞在輸卵管局部浸潤情況。感染后CD3+CD4+和CD3+CD8α+在輸卵管局部逐漸增多。在膨大部黏膜上皮基部和固有層,CD3+CD8α+淋巴細胞浸潤情況比CD3+CD4+更為明顯。綜上所述,本研究證實H9N2 AIV感染蛋雞后病毒能在輸卵管組織存活復制,且引起輸卵管發(fā)生組織凋亡和其他病理損傷。此外,H9N2 AIV能夠引起蛋雞輸卵管免疫相關基因的表達以及CD3+CD4+和CD3+CD8α+淋巴細胞的浸潤。為了解蛋雞感染H9N2產生的臨床癥狀和產蛋機能失調奠定了理論基礎。
[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.

【學位授予單位】：西北農林科技大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S858.31

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本文編號：1443282