【作者】 刘洋；

【导师】 满朝来；

【作者基本信息】 哈尔滨师范大学 ， 生物化学与分子生物学， 2021， 硕士

【摘要】 miRNA是一类参与免疫调控的重要影响因子,循环miRNA在反映机体状态和成为分子标记物方面具有重要的研究价值。目前,关于循环miRNA在免疫应答和应激性免疫抑制中的动态表达规律未见报道。本研究以鸡为模式动物,利用新城疫病毒(NDV)La Sota疫苗和地塞米松(Dex)分别制备免疫应答模型和应激性免疫抑制模型,采用荧光定量颈环RT-PCR技术,对机体不同免疫状态下的15种NDV相关的血清循环miRNA(miR-124、miR-19b、miR-126、miR-199、miR-375、miR-30、miR-122、miR-22、miR-20、miR-34、miR-200b、miR-29b、miR-451、miR-198和miR-31)进行表达规律分析,选取3种差异表达的miRNA(miR-375,miR-19b和miR-20)进一步分析其在组织中的时空表达规律,并用生物信息学预测其分子机制。主要研究结果如下:正常雏鸡NDV疫苗免疫应答过程中,免疫后的2-3天(dpi)和14-28dpi是血清循环miRNA响应NDV免疫应答的关键时期;应激性免疫抑制雏鸡中,2dpi和14dpi～28dpi是Dex诱导的大部分循环miRNA明显上调的时间点,7dpi的相对表达活性都较低;而在应激性免疫抑制雏鸡进行NDV疫苗免疫过程中,血清循环miRNA的差异表达时间点主要集中在2dpi和5dpi,且7dpi-21dpi的相对表达活性都较低。应激性免疫抑制能够使血清循环miRNA对NDV的响应关键时间点由正常机体的2dpi-3dpi和14dpi-28dpi转变为2dpi和5dpi,并抑制血清循环miRNA在NDV疫苗特异性免疫应答(7dpi-21dpi)的表达。正常雏鸡NDV疫苗免疫应答过程中,miR-375参与法氏囊(2dpi和21dpi),心、肺和胸腺(2dpi),脾和血细胞(14dpi)中的免疫调控过程,且法氏囊在2dpi、5dpi、14dpi和21dpi时可能是影响血清循环miR-375的表达水平的关键器官;应激性免疫抑制雏鸡中,miR-375是胸腺和法氏囊(5dpi),脾脏和腺胃(14dpi),血细胞(5dpi和14dpi)参与应激性免疫抑制调控的重要影响因子,2dpi的法氏囊可能是影响血清miR-375变化的关键器官;应激性免疫抑制雏鸡进行NDV疫苗免疫过程中,心、胸腺和法氏囊(2dpi),小肠、结肠、脾脏和血细胞(14dpi)是免疫抑制条件下miR-375积极响应NDV免疫应答的器官,且2dpi的法氏囊可能是影响血清miR-375变化的关键器官。生物信息学分析表明,miR-375可能主要通过调控QKI、ELAVL4、PAX6、RPBPJ、TCPK、TCF12、TCCH2、KAF4、NR5A2、PIAS1形成的蛋白网络参与免疫调控过程。正常雏鸡NDV疫苗免疫应答过程中,miR-19b通过结肠和腺胃(2dpi)、法氏囊(2dpi和21dpi)参与NDV免疫应答过程;应激性免疫抑制雏鸡中,miR-19b通过参与心脏和法氏囊(2dpi)、胸腺(5dpi)、血细胞(14dpi)、结肠(21dpi)的免疫调控来应答Dex引起的应激性免疫抑制。应激性免疫抑制雏鸡进行NDV疫苗免疫过程中,2dpi的法氏囊和腺胃、5dpi的心、14dpi的小肠和结肠是miR-19b此过程中关键的免疫应答器官。生物信息学分析表明,miR-19b可能主要通过调控RNF11、FBXO30、ITCH、KLHL11、SMURF1、SMURF2、CBLB、LONRF1、MYLIP、HECW2、KBTBD8、WWP1形成的蛋白网络影响多种免疫过程。正常雏鸡NDV疫苗免疫应答过程中,miR-20可能通过结肠(2dpi)和法氏囊(21dpi)发挥着重要的免疫调节功能;应激性免疫抑制雏鸡中,miR-20通过心、结肠和盲肠扁桃体(2dpi)、胸腺(5dpi)、血细胞(14dpi)积极参与应激性免疫抑制过程;应激性免疫抑制雏鸡进行NDV疫苗免疫过程中,2dpi的结肠、14dpi的小肠和结肠是miR-20参与此过程的关键的免疫应答器官。生物信息学分析表明,miR-20可能主要通过调控RNF6、NEDD4L、FBXW11、FBXO30、HECTD2、MKRN1、TRIM36、MYLIP、FBXL5蛋白网络介导的泛素化过程参与机体免疫功能调控。本研究对不同免疫状态下15种NDV相关血清循环miRNA的表达特性进行分析,发现应激性免疫抑制能够改变NDV疫苗免疫通路中miRNA的时空表达特性,而且预测出3种关键miRNA参与免疫调控的可能作用机制,结果表明miRNA是影响免疫应答和应激性免疫抑制的重要调节因子。本研究为深入研究应激性免疫抑制与免疫反应的分子作用机制和开发以miRNA为靶标的家禽免疫状态辅助检测技术提供理论参考。更多还原

【Abstract】 miRNAs are a kind of important factors in immune regulation,and circulating miRNAs are of vital significance in reflecting the immune state of the body and hence have the potential to become molecular markers.However,the dynamic expression of circulating miRNA in immune response and stress-induced immunosuppression has not been fully clarified.In this study,chickens were used as model animals,and immune response and stress-induced immunosuppressive chicken were prepared using Newcastle disease virus(NDV)(La Sota vaccine)and Dexamethasone(Dex),respectively.And expression activities of 15 NDV related circulating miRNAs(miR-124、miR-19b、miR-126、miR-199、miR-375、miR-30、miR-122、miR-22、miR-20、miR-34、miR-200b、miR-29b、miR-451、miR-198 and miR-31)in different immune states were identified by using Quantitative stem-loop RT-PCR.Three differentially expressed miRNAs(miR-375,miR-19 b and miR-20)were selected to analyze tissue spatiotemporal expression patterns and their molecular mechanisms were subsequently predicted by bioinformatics methods.Major results are as follows:In NDV vaccine induced healthy chicken,2-3dpi(day post immunization)and14-28 dpi were the key periods that serum circulating miRNAs response.In Dex induced immunosuppressive chicken,2dpi and 14dpi-28 dpi were the time points when most of the circulating miRNAs were up-regulated,and the expression activities of 7dpi was relative low.In NDV vaccine induced immunosuppressive chicken,the time points of differentially expressed serum circulating miRNAs were mainly concentrated in 2dpi and 5dpi and expression activities of 7dpi-21 dpi were relative low.Stress-induced immunosuppression changed the critical time points of serum circulating miRNA to NDV-from 2-3dpi and 14-28 dpi to 2dpi and 5dpi,and mainly inhibited the response of circulating miRNAs in specific immune phase of NDV vaccine immune response(7dpi-21dpi).In NDV vaccine induced healthy chicken,miR-375 was actively involved in the immune regulation of bursa of Fabricius(2dpi and 21dpi),heart,lung and thymus(2dpi),spleen and blood cells(14dpi),and 2dpi,4dpi,14 dpi and 21dpi’s the bursa of Fabricius might be the key tissues affecting the expression level of serum circulating miR-375.In Dex-induced immunosuppressive chicken,miR-375 was an important regulator of thymus and bursa of Fabricius(5dpi),spleen and glandular stomach(14dpi),blood cells(5dpi and 14dpi)which involved in stress-induced immunosuppression,and 2dpi’s bursa of Fabricius in the process may be the most probable organ affecting the expression level of serum circulating miR-375.In NDV vaccine induced immunosuppressive chicken,heart,thymus and bursa of Fabricius(2dpi),small intestine,colon,spleen and blood cells(14dpi)were the organs that miR-375 responded to NDV under immunosuppression,and 2dpi’s the bursa of Fabricius in the process might be the key organ affecting serum circulating miR-375.Bioinformatics analysis predicted that miR-375 was involved in a variety of immune regulation processes by regulating the protein network formed by QKI,ELAVL4,PAX6,RPBPJ,TCPK,TCF12,TCCH2,KAF4,NR5A2 and PIAS1.In NDV vaccine induced healthy chicken,miR-19 b participated in NDV vaccine immune response through colon and glandular stomach(2dpi),bursa of Fabricius(2dpi and 21dpi).In Dex-induced immunosuppressive chicken,miR-19 b was involved in the immune regulation of heart and bursa of Fabricius(2dpi),thymus(5dpi),blood cells(14dpi)and colon(21dpi)to respond to stress-induced immunosuppression.In NDV vaccine induced immunosuppressive chicken,the bursa and glandular stomach of 2dpi,heart of 5dpi,small intestine and colon of 14 dpi were the key immune response organs of miR-19 b.Bioinformatics analysis predicted that miR-19 b was involved in a variety of immune regulation processes by regulating the protein network formed by RNF11,FBXO30,TCH,KLHL11,SMURF1,SMURF2,CBLB,LONRF1,MYLIP,HECW2,KBTBD8 and WWP1.In NDV vaccine induced healthy chicken,miR-20 might play the important role in immune regulation through colon(2dpi)and bursa of Fabricius(21dpi).In Dex induced immunosuppressive chicken,miR-20 actively participated in the process of stress-induced immunosuppression through heart,colon and cecal tonsil(2dpi),thymus(5dpi)and blood cells(14dpi).In NDV vaccine induced immunosuppressive chicken,colon(2dpi),small intestine and colon(14dpi)were the key immune tissues of miR-20 involved in this process.Bioinformatics analysis predicted that miR-20 was involved in a variety of immune regulation processes by regulating the protein network formed by RNF6,NEDD4 L,FBXW11,FBXO30,HECTD2,MKRN1,TRIM36,MYLIP and FBXL5.In this study,we analyzed the expression characteristics of 15 NDV-related serum circulating miRNAs in different immune states,and it was found that stress immunosuppression could change the spatiotemporal expression of miRNA in NDV vaccine immune pathway,the possible mechanisms of 3 key miRNAs involved in NDV vaccine immune regulation were analyzed.These findings indicate that circulating miRNA were important regulators of immune response and stress-induced immunosuppression and provide theoretical and practical reference for further study on the molecular mechanism of stress-induced immunosuppression and immune response.更多还原