microRNA-155在缺硒性肉鸡淋巴细胞凋亡中作用机制的研究

发布时间：2018-03-07 00:30

本文选题：肉鸡　切入点：硒　出处：《东北农业大学》2017年博士论文　论文类型：学位论文

【摘要】：硒是动物机体中不可或缺的营养元素,参与多种生物学功能。硒主要参与动物机体内的神经生物学、肌肉代谢、生殖、氧化还原反应、延缓衰老、病毒感染等多种生物学过程。硒对免疫系统具有多方面的重要作用。硒主要通过保持免疫细胞膜的完整性,维持免疫系统的生理功能,从而发挥其增强机体免疫力,预防疾病等重要作用。以前的研究报道缺硒能够导致人、鼠、蛋鸡和猪等多种动物的免疫组织损伤或免疫反应异常,可诱导免疫细胞活性降低,抑制机体固有免疫、细胞免疫和体液免疫功能。细胞凋亡是免疫抑制机制之一,研究报道硒能诱导免疫细胞凋亡的发生。此外,研究表明硒的水平也能改变细胞内micro RNAs(mi RNA)表达谱及其相关基因m RNAs表达,从而影响细胞的正常生理功能。Mi RNA-155是一种多功能的mi RNA,也是目前发现的靶基因最多的mi RNA之一。Mi RNA-155在免疫系统中的作用尤为重要,不但调节免疫细胞的发育和分化过程,并且影响免疫细胞的功能。目前对mi RNA-155在缺硒肉鸡免疫组织细胞凋亡中的作用尚不清楚。本试验在复制缺硒肉鸡免疫组织凋亡模型和淋巴细胞mi RNA-155过表达/敲低模型的基础上,探究mi RNA-155与免疫组织细胞凋亡的相关性,明确mi RNA-155在鸡缺硒性淋巴细胞凋亡中的信号转导通路。结果表明:1. 使用低硒(0.03 mg/kg)日粮饲喂雏肉鸡30 d后,发现肉鸡免疫功能降低,同时在脾脏,胸腺和法氏囊组织超微结构和组织病理结构学中观察到免疫细胞发生了凋亡现象,证实成功复制了缺硒肉鸡免疫组织细胞凋亡模型;2.通过检测低硒肉鸡免疫组织内抗氧化酶相关基因(Gpx和Mn SOD)m RNA和凋亡相关基因(JNK、Bcl-2、Bax、Bak、Cyt-c、caspase 9、caspase 3)m RNA及蛋白表达,结果显示缺硒诱导免疫组织发生氧化应激性凋亡。3.脾脏组织mi RNA组学结果发现检测的缺硒组内657个mi RNAs中,有205个mi RNAs表达升高,181个mi RNAs表达降低,mi RNA-155的表达水平也降低;q PCR结果显示缺硒组mi RNA-155的表达丰度降低至对照组的43%。结果证实缺硒可改变脾脏组织内mi RNAs表达谱,并显著抑制了mi RNA-155的表达;4.应用细胞转染法,将合成的mi RNA-155核酸链转染至原代培养的鸡淋巴细胞内,结果显示过表达组淋巴细胞内mi RNA-155表达显著升高了7.9倍,敲低组淋巴细胞内mi RNA-155表达降低至对照组的47%。表明原代培养鸡淋巴细胞mi RNA-155过表达/敲低模型建立成功;5.将预测靶基因TNFRSF1B(野生型及突变型)的重组质粒与mi RNA-155共转染入培养的鸡淋巴细胞,结合双荧光素酶基因报告试验、q PCR和Western bloting等试验结果证实在淋巴细胞内TNFRSF1B确实是mi RNA-155的靶基因之一;6.使用H_2O_2处理mi RNA-155过表达/敲低的淋巴细胞,通过AO-EB、Tunel、抗氧化酶相关基因(Gpx和Mn SOD)、凋亡相关基因(JNK、Bcl-2、Bak、Bak、Cyt-c、caspase9、caspase3)的检测,结果揭示mi RNA-155在鸡淋巴细胞内能够通过靶向TNFRSF1B调控H_2O_2诱导的氧化应激性凋亡;7.通过检测肉鸡免疫组织内Ca~(2+)稳态相关基因(Ry R1、Ry R3和SERCA1s),内质网应激相关基因(GRP78、GRP94、IRE1、e IF2α、ATF6和caspase12)的表达,结果显示缺硒诱导肉鸡免疫组织发生了由Ca~(2+)失调引起的内质网应激。而结合体外试验,结果显示mi RNA-155有效缓解了淋巴细胞内质网应激的发生。综上所述,缺硒诱导mi RNA-155的低表达能通过负调控TNFRSF1B,进一步促进了氧化应激性细胞凋亡。并且证明mi RNA-155能影响缺硒肉鸡免疫组织细胞内质网应激和免疫功能。本试验为进一步研究mi RNA-155与缺硒之间的作用机制奠定了基础,为防治缺硒性免疫功能障碍提供理论依据,也为比较医学提供借鉴。
[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.

【学位授予单位】：东北农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S858.31

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