microRNA-155在鸡缺硒性脾淋巴细胞凋亡中的作用机制研究

Excessive apoptosis in spleen lymphocyte is the main mechanism of immunosuppressive induced by selenium deficiency in chicken. MicroRNA-155 involved in regulating the immune organ differentiation, development and apoptosis. However, the role of microRNA-155 in apoptosis in chicken spleen lymphocyte still unclear.The main reason of immunosuppression induced by selenium deficiency in chicken is spleen lymphocyte apoptosis. MicroRNA-155 is the major one of microRNA in immune system, which it not only plays an important role in the development and differentiation of immune organ, but also influence cell apoptosis through the special signal transduction pathways. Base on the cases model of chicken splenic lymphocyte apoptosis induced by selenium deficiency in vitro, using real-time quantitative PCR, western blot, transmission electron microscope and omics technology methods to detect the transcriptome, proteomics and the level of microRNA-155. Combined with bioinformatics technology, screening the target genes of microRNA-155 and the possible signal pathway in chicken splenic lymphocytes induced by selenium deficiency. On this basis, modeling microRNA-155 knockdown and overexpression splenic lymphocytes in vitro, using flow cytometry, laser confocal microscopy, real-time quantitative PCR and western blot methods to detect the cell apoptosis, calcium homeostasis and the expression of target genes. The implementation of this project will contribute to a better understanding of the mechanism of microRNA-155 on the signal transduction in cell apoptosis in chicken splenic lymphocytes. The result of this project will provide not only a basis of prevention and treatment of immune function barrier induced by selenium deficiency, but also a theoretical reference for comparative medicine.

脾淋巴细胞过度凋亡是鸡缺硒性免疫抑制机制之一，microRNA-155参与调控免疫器官的分化、发育和细胞凋亡，但其在鸡缺硒性免疫器官脾脏的细胞凋亡中的作用尚不清楚。本课题拟在建立鸡缺硒性脾脏淋巴细胞凋亡模型的基础上，应用实时荧光定量PCR、免疫印迹、透射电镜和组学技术等，检测脾脏转录组、蛋白组和microRNA-155的表达，结合生物信息学技术，筛选microRNA-155的靶基因及其在鸡缺硒性脾脏细胞凋亡发生中的可能信号转导通路，在此基础上，体外培养鸡脾脏淋巴细胞，建立microRNA-155抑制与过表达模型、其靶基因敲低和过表达模型，应用流式细胞术、激光共聚焦、实时定量荧光PCR和免疫印迹等方法，检测细胞凋亡、钙稳态、靶基因表达等，阐明microRNA-155在鸡缺硒性脾脏细胞凋亡发生中的信号转导通路，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。

硒缺乏会导致免疫细胞凋亡的发生，MiRNA-155是一种多功能的miRNA，在免疫系统中的作用尤为重要，不仅调节免疫细胞的发育和分化过程，还影响免疫细胞的功能。本研究在复制缺硒肉鸡免疫组织凋亡模型和淋巴细胞miRNA-155过表达/敲低模型的基础上，探究miRNA-155与免疫组织细胞凋亡的相关性，明确miRNA-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. 缺硒诱导免疫组织发生氧化应激性凋亡。2. 脾脏组织miRNA组学结果发现检测的缺硒组内657个miRNAs中，有205个miRNAs表达升高，181个miRNAs表达降低，其中miRNA-155的表达水平也降低；缺硒组miRNA-155的表达丰度降低至对照组的43 %，这些结果说明缺硒可改变脾脏组织内miRNAs表达谱，并显著抑制了miRNA-155的表达；3. 鸡淋巴细胞miRNA-155过表达/敲低模型建立成功；4.结合双荧光素酶基因报告试验、qPCR和Western bloting等试验结果证实在淋巴细胞内TNFRSF1B确实是miRNA-155的靶基因之一；5.miRNA-155在鸡淋巴细胞内能够通过靶向TNFRSF1B调控H2O2诱导的氧化应激性凋亡；6.通过检测肉鸡免疫组织内Ca2+稳态相关基因（RyR1、RyR3和SERCA1s），内质网应激相关基因（GRP78、GRP94、IRE1、eIF2α、ATF6和caspase12）的表达，结果显示缺硒诱导肉鸡免疫组织发生了由Ca2+失调引起的内质网应激。结合体外试验，结果显示miRNA-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miRNA-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miRNA-155能影响缺硒肉鸡免疫组织细胞内质网应激和免疫功能。此外，还进行了日粮硒缺乏对鸡肝脏蛋白质组学及自噬变化的影响，低硒日粮对肉鸡免疫器官中硒蛋白与细胞因子表达的影响、microRNA-193b-3p通过靶向MAML1调节硒缺乏型肉鸡肝细胞凋亡、miR-128-1-5p靶向CADM1在缺硒性肉鸡VECs中紧密连接作用机制的探讨等，本研究为进一步研究miRNA与缺硒之间的作用机制奠定了基础，为防治缺硒性损伤提供理论依据，也为比较医学提供借鉴。