本文選題：肉雞　切入點：硒　出處：《東北農(nóng)業(yè)大學》2017年博士論文　論文類型：學位論文

【摘要】：硒是動物機體中不可或缺的營養(yǎng)元素,參與多種生物學功能。硒主要參與動物機體內的神經(jīng)生物學、肌肉代謝、生殖、氧化還原反應、延緩衰老、病毒感染等多種生物學過程。硒對免疫系統(tǒng)具有多方面的重要作用。硒主要通過保持免疫細胞膜的完整性,維持免疫系統(tǒng)的生理功能,從而發(fā)揮其增強機體免疫力,預防疾病等重要作用。以前的研究報道缺硒能夠導致人、鼠、蛋雞和豬等多種動物的免疫組織損傷或免疫反應異常,可誘導免疫細胞活性降低,抑制機體固有免疫、細胞免疫和體液免疫功能。細胞凋亡是免疫抑制機制之一,研究報道硒能誘導免疫細胞凋亡的發(fā)生。此外,研究表明硒的水平也能改變細胞內micro RNAs(mi RNA)表達譜及其相關基因m RNAs表達,從而影響細胞的正常生理功能。Mi RNA-155是一種多功能的mi RNA,也是目前發(fā)現(xiàn)的靶基因最多的mi RNA之一。Mi RNA-155在免疫系統(tǒng)中的作用尤為重要,不但調節(jié)免疫細胞的發(fā)育和分化過程,并且影響免疫細胞的功能。目前對mi RNA-155在缺硒肉雞免疫組織細胞凋亡中的作用尚不清楚。本試驗在復制缺硒肉雞免疫組織凋亡模型和淋巴細胞mi RNA-155過表達/敲低模型的基礎上,探究mi RNA-155與免疫組織細胞凋亡的相關性,明確mi RNA-155在雞缺硒性淋巴細胞凋亡中的信號轉導通路。結果表明:1. 使用低硒(0.03 mg/kg)日糧飼喂雛肉雞30 d后,發(fā)現(xiàn)肉雞免疫功能降低,同時在脾臟,胸腺和法氏囊組織超微結構和組織病理結構學中觀察到免疫細胞發(fā)生了凋亡現(xiàn)象,證實成功復制了缺硒肉雞免疫組織細胞凋亡模型;2.通過檢測低硒肉雞免疫組織內抗氧化酶相關基因(Gpx和Mn SOD)m RNA和凋亡相關基因(JNK、Bcl-2、Bax、Bak、Cyt-c、caspase 9、caspase 3)m RNA及蛋白表達,結果顯示缺硒誘導免疫組織發(fā)生氧化應激性凋亡。3.脾臟組織mi RNA組學結果發(fā)現(xiàn)檢測的缺硒組內657個mi RNAs中,有205個mi RNAs表達升高,181個mi RNAs表達降低,mi RNA-155的表達水平也降低;q PCR結果顯示缺硒組mi RNA-155的表達豐度降低至對照組的43%。結果證實缺硒可改變脾臟組織內mi RNAs表達譜,并顯著抑制了mi RNA-155的表達;4.應用細胞轉染法,將合成的mi RNA-155核酸鏈轉染至原代培養(yǎng)的雞淋巴細胞內,結果顯示過表達組淋巴細胞內mi RNA-155表達顯著升高了7.9倍,敲低組淋巴細胞內mi RNA-155表達降低至對照組的47%。表明原代培養(yǎng)雞淋巴細胞mi RNA-155過表達/敲低模型建立成功;5.將預測靶基因TNFRSF1B(野生型及突變型)的重組質粒與mi RNA-155共轉染入培養(yǎng)的雞淋巴細胞,結合雙熒光素酶基因報告試驗、q PCR和Western bloting等試驗結果證實在淋巴細胞內TNFRSF1B確實是mi RNA-155的靶基因之一;6.使用H_2O_2處理mi RNA-155過表達/敲低的淋巴細胞,通過AO-EB、Tunel、抗氧化酶相關基因(Gpx和Mn SOD)、凋亡相關基因(JNK、Bcl-2、Bak、Bak、Cyt-c、caspase9、caspase3)的檢測,結果揭示mi RNA-155在雞淋巴細胞內能夠通過靶向TNFRSF1B調控H_2O_2誘導的氧化應激性凋亡;7.通過檢測肉雞免疫組織內Ca~(2+)穩(wěn)態(tài)相關基因(Ry R1、Ry R3和SERCA1s),內質網(wǎng)應激相關基因(GRP78、GRP94、IRE1、e IF2α、ATF6和caspase12)的表達,結果顯示缺硒誘導肉雞免疫組織發(fā)生了由Ca~(2+)失調引起的內質網(wǎng)應激。而結合體外試驗,結果顯示mi RNA-155有效緩解了淋巴細胞內質網(wǎng)應激的發(fā)生。綜上所述,缺硒誘導mi RNA-155的低表達能通過負調控TNFRSF1B,進一步促進了氧化應激性細胞凋亡。并且證明mi RNA-155能影響缺硒肉雞免疫組織細胞內質網(wǎng)應激和免疫功能。本試驗為進一步研究mi RNA-155與缺硒之間的作用機制奠定了基礎,為防治缺硒性免疫功能障礙提供理論依據(jù),也為比較醫(yī)學提供借鑒。
[Abstract]:Selenium is an essential nutrient element in animal body, participate in a variety of biological functions. The neurobiology, selenium mainly involved in animal organism muscle metabolism, reproduction, redox reaction, aging, virus infection and other biological processes. Selenium plays an important role in many aspects of the immune system. Selenium mainly by maintaining immune cell membrane the maintenance of physiological function of the immune system, which play an important role to enhance immunity, prevent disease. Previous studies reported selenium deficiency can lead to human, rat, abnormal immune tissue damage or immune reaction of the chicken and pig and other animal, can induce the immune cell activity decreased, inhibiting the innate immune and humoral immune. The immune function of cells. Cell apoptosis is one of mechanisms of immunosuppression, research reports of selenium can induce immune cell apoptosis. In addition, studies have shown that selenium level Can change the intracellular micro RNAs (MI RNA) expression and M gene expression of RNAs, thus affecting the physiological function of.Mi RNA-155 cells mi RNA is a versatile, target gene is also found that the most mi RNA of.Mi RNA-155 in the immune system is particularly important, development and differentiation not only the regulation of immune cells, and affect the function of immune cells. The role of MI RNA-155 in selenium deficient immune tissues of broilers in apoptosis is still unclear. This experiment is based on replication deficient immune tissue selenium broiler apoptosis model and lymphocyte mi RNA-155 overexpression / knockdown model, correlation between MI RNA-155 and immunohistochemical study cell apoptosis, clear mi RNA-155 in selenium deficiency in Chicken Lymphocyte Apoptosis in signal transduction pathway. The results showed that: 1. (0.03 mg/kg) with low selenium diet of broiler chicks after 30 d in Broilers At the same time, decreased immune function, spleen, pathological structure and ultrastructure of thymus and bursa tissue histology observed in immune cell apoptosis phenomenon, confirmed successfully replicated selenium deficient immune tissues of broilers apoptosis model; 2. through the detection of low selenium chicken immune tissue antioxidant enzyme genes (Gpx and Mn SOD m) RNA and apoptosis related genes (JNK, Bcl-2, Bax, Bak, Cyt-c, caspase 9, caspase 3) expression of M protein and RNA, results showed that oxidative stress induced apoptosis of.3. spleen tissue in MI group RNA study found that 657 mi RNAs selenium deficiency group detection in selenium deficiency induced by immune organization, 205 Mi increased the expression of RNAs 181, MI RNAs expression decreased, MI expression level of RNA-155 decreased; Q PCR results showed that the expression abundance of MI RNA-155 in selenium deficiency group decreased to the control group 43%. showed that selenium deficiency can change the expression of MI RNAs in spleen, and significantly The inhibited the expression of MI RNA-155; 4. by cell transfection method, the synthesis of MI RNA-155 nucleic acid chain was transfected into primary cultured chicken lymphocytes, results showed that overexpression of MI in lymphocytes of RNA-155 group significantly increased 7.9 times, knocking down the expression of RNA-155 MI in the group of lymphocytes decreased to 47%. of the control group showed that primary culture of chicken Mi lymphocytes RNA-155 expression / knockdown model was established successfully; 5. predicted target gene TNFRSF1B (wild type and mutant) and Mi recombinant plasmid RNA-155 were co transfected into cultured chicken lymphocytes with dual luciferase gene report test, Q PCR and Western bloting results confirmed the lymphocyte TNFRSF1B is indeed one of the the target gene mi RNA-155; 6. Mi using H_2O_2 RNA-155 overexpression / knockdown of lymphocytes by AO-EB, Tunel, antioxidant enzyme related genes (Gpx and Mn SOD) and apoptosis related matrix Because of (JNK, Bcl-2, Bak, Bak, Cyt-c, caspase9, Caspase3) detection, the results reveal that MI RNA-155 can be targeted by TNFRSF1B regulation of H_2O_2 induced apoptosis of lymphocytes in oxidative stress in chicken; 7. by immunohistochemical detection of Ca~ in broilers (2+) homeostasis related genes (Ry R1, Ry R3 and SERCA1s), endoplasmic reticulum stress related genes (GRP78, GRP94, IRE1, e, IF2 alpha, ATF6 and caspase12) expression results showed that selenium deficiency induced broiler immune tissue changed from Ca~ (2+) caused by the imbalance of endoplasmic reticulum stress. And combined with in vitro test, the results showed that MI RNA-155 lymphocytes effectively alleviate the endoplasmic reticulum stress occurs. In conclusion, the low expression of MI RNA-155 induced by selenium deficiency through the negative regulation of TNFRSF1B, to further promote the apoptosis of cells. Oxidative stress and that MI RNA-155 can influence the selenium deficiency immune tissues of broilers endoplasmic reticulum stress and immune function. The test for Further research on the mechanism of MI RNA-155 and selenium deficiency will provide a theoretical basis for prevention and treatment of selenium deficiency immune dysfunction, and provide reference for comparative medicine.

【學位授予單位】：東北農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S858.31

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