Toll样受体信号传导通路介导硒缺乏雏鸡免疫损伤机制研究

Chicken selenium deficiency can cause host immune dysfunction, however, it is unclear that the key molecules and signal pathway which mediates, adjusts or amplifies the damage of cells, tissues and even organs during this process. Our previous reports have showed that some endogenous substance recognizing TLR and involved in the immune damage in selenium deficiency. In the project, we will detect the dynamitic changes of related factors (including 10 kinds of chTLRs, MyD88, TRIF and NF-κB) and cytokines (IFN-γ, TNF-α, IL-2 and IL-6) in the signal transduction pathway of immune organs at different times of chicken selenium deficiency by real time PCR, Western Blot, and other techniques, and illustrate preliminarily the kind(s) toll-like receptor mediating the immune injury. After that, we will silence the high level expression of chTLRs and adaptor molecular of MyD88 or TRIF to confirm the chTLRs and key molecules in signal transduction pathway related to immune injury. This will illuminate the immune dysfunction mechanism of chicken selenium deficiency at the molecular level of TLR signal transduction pathway and provide important scientific basis for effective prevention and treatment of selenium deficiency in poultry.

雏鸡硒缺乏可引起免疫器官机能降低，但免疫损伤过程中介导、调节或者放大细胞、组织甚至器官损伤的关键分子和信号通路还不清楚。我们前期研究发现，雏鸡硒缺乏过程存在TLR识别的内源性物质并影响免疫损伤，故本研究将TLR信号传导通路与雏鸡硒缺乏免疫损伤相关联，通过实时荧光定量PCR和Western Blot等方法检测雏鸡硒缺乏不同时期免疫器官chTLRs信号传导通路相关因子（包括10种chTLR、MyD88 、TRIF及NF-κB）及细胞因子（IFN-γ、TNF-α、IL-2和IL-6）活化变化，以初步分析介导免疫损伤的Toll样受体种类；进而干扰沉默高表达chTLRs及衔接蛋白分子MyD88 或 TRIF 基因，以确定与免疫损伤相关chTLRs及信号传导通路关键蛋白分子；该研究从TLR信号传导通路角度在分子水平阐述雏鸡硒缺乏免疫损伤机制，为有效防治禽类硒缺乏病提供重要的科学依据。

硒缺乏可导致雏鸡胸腺、脾脏和法氏囊等免疫器官出现淋巴细胞数量减少、结构和功能紊乱等病理变化。寻求免疫器官损伤过程中介导、调节或者放大细胞、组织甚至器官损伤的关键分子和信号通路显得十分重要。我们前期研究发现，硒缺乏可使雏鸡体内产生TLR识别的内源性物质。但关于TLR是否参与Se缺乏雏鸡免疫器官损伤的研究尚不清楚。本研究复制硒缺乏雏鸡模型，制作石蜡切片并进行HE染色。结果显示到免疫器官均表现为淋巴细胞数量减少，细胞核浓缩、碎裂等坏死行变化。这表明硒缺乏可导致雏鸡胸腺、脾脏和法氏囊发生病理损伤。透射电镜被用来观察胸腺、脾脏和法氏囊超微结构。结果发现其细胞均表现为核内染色质边聚，贴附于核膜下；胞浆内线粒体肿胀，嵴紊乱，部分结构消失，甚至出现空泡化；内质网排列紊乱，网池增宽，融合成空泡状，有的出现了断裂。我们设计合成8种ChTLRs、MyD88、TRIF、NF-κB及4种细胞因子（IL-2、IL-6、TNF-α和IFN-γ）引物，用实时荧光定量PCR检测缺硒组和对照组胸腺、脾脏、法氏囊中15种基因mRNA表达水平，用Western blot 方法检测TLR4的蛋白表达水平。结果发现硒缺乏组雏鸡脾脏内TLR4和TRIF表达水平升高，法氏囊内TLR15和MyD88表达水平升高。这表明TLR4通过TRIF通路参与了脾脏的免疫损伤，TLR15通过MyD88通路参与了法氏囊的免疫损伤。体外培养DT40和HD11细胞，建立细胞低硒培养体系，检测8种ChTLRs、MyD88、TRIF、NF-κB及4种细胞因子mRNA表达水平，与体内检测结果一致。故合成TLR4、TLR15、MyD88和TRIF的小干扰RNA及过表达质粒，进行下游细胞因子相关基因的检测。结果显示TLR4和TLR15在硒缺乏引起的免疫细胞损伤中起着重要作用。综上结果表明，硒缺乏激活了TLR信号传导通路，且该信号传到通路参与并介导了雏鸡免疫器官损伤。