基于血液转录组的大熊猫免疫相关基因表达谱研究

The giant panda has been greatly threatened by diseases and the characteristic of individual immunity is closely associated with their disease occurrence and healing. Thus the investigations on the immunity characteristic of giant panda are important for this species conservation. In this study, the blood transcriptome data will be used to profile the expressions of immune-related genes of giant panda with its several important growth and female-typical physiological periods, receiving vaccination against the canine distemper virus (CDV) and stimuli by various field environmental factors in the wildness training of giant panda. Our objectives are: (1) to understand the immunity characteristic of several important growth periods and female-typical physiological periods of giant panda, assess their immunity situations and disease risks, and give service to developing specific means of disease prevention for captive giant panda; (2) to provide insight into the pattern of immune response to the complex field environmental factors and its dynamic change in the wildness training of giant panda. Through the comparison to that of captive giant pandas, to screen out some molecular markers, which can be used to evaluate and monitor the immunity situations of trained giant pandas; (3) to pinpoint the immune pathways and expression characteristics of immune-related genes activated by vaccination against CDV and search the clues about the pathogenesis and treatment of giant panda canine distemper. Additionally, in the study, SNPs of immune-related genes will be identified and the associations between them and expression of the corresponding genes will be also analyzed. This may provide important resource for these studies of immune-related disease pathogenesis.

疾病严重威胁大熊猫的种群安全，而疾病发生和治疗与大熊猫自身免疫特性密切相关，因此开展大熊猫免疫基础研究对于大熊猫的保护具有重要意义。本研究基于高通量血液转录组分析，建立大熊猫重要生长阶段和母兽几个特殊生理时期以及野放培训复杂环境下和犬瘟热疫苗攻击后免疫相关基因的表达谱，掌握圈养大熊猫重要生长阶段和母兽几个特殊生理时期的免疫特性，评价免疫状况和不同类疾病发生的风险，为建立圈养大熊猫有针对性的疾病预防措施提供理论依据；探明野放培训大熊猫免疫应答策略和培训期间的动态变化，通过与圈养大熊猫比较分析，筛选野化培训大熊猫免疫评价的分子标记，为野化培训大熊猫免疫状态的监测和评价奠定理论基础、建议评价指标；掌握犬瘟热疫苗攻击后大熊猫免疫相关基因的表达特性，探索其免疫应答的分子机制及该病的治疗线索。研究还将鉴定免疫相关基因的SNP位点，探讨其与相关基因表达量的关系，为相关免疫疾病发生机制的研究提供重要资源。

基于血液转录组分析，我们获得了大熊猫免疫相关基因的表达概况，鉴定了不同免疫类型的15个免疫通路，涉及到免疫相关基因2207个转录子。主要组织相容性复合体（MHC）家族中，Aime-128 MHCⅠ类基因的表达水平远高于其他基因，暗示Aime-128 MHCⅠ类基因在圈养大熊猫的免疫中可能发挥关键作用。我们在血液转录组中鉴定了11890个SNP位点，其中CDS上有590个，他们涉及267个基因，功能富集到天然杀伤细胞介导细胞毒性，B细胞受体信号通路，抗原过程和呈现等10条免疫相关的通路，表明这些SNP位点与大熊猫免疫功能的发挥密切相关。我们也鉴定了210个年龄相关的免疫基因，老年个体上调基因大都与先天性免疫相关，相反，下调基因与适应性免疫相关，表明随着老年化，大熊猫先天性免疫增强、适应性免疫降低。ISG15、STAT1、IRF7和DDX58等几个hub基因，在年龄相关的免疫改变中可能发挥关键作用。.发情期受体识别和炎症反应相关的基因表达量上调，而参与T细胞激活和增殖的基因显著下调，表明发情期雌性大熊猫先天性免疫增强，而适应性免疫减弱，发情期需经历局部组织并伴随强烈的炎症反应。妊娠早期，炎症反应、先天免疫功能以及NK细胞活性受到抑制，大熊猫可能通过牺牲一定的免疫功能以实现免疫耐受。妊娠晚期，NK细胞功能依旧受到抑制，NOD样介导的先天免疫增强，母体增强对病原体的防御以保护胎儿。相比圈养大熊猫，野化培训大熊猫有239个基因上调、61基因下调，功能富集表明野放培训大熊猫有提高的先天性免疫和适应性免疫，PRF1、TBX21等hub基因可以作为免疫适应监测的候选基因。.项目观察了大熊猫响应CDV疫苗的免疫应答，与先前血清学抗体研究结果相似，疫苗主要激活了大熊猫的先天性免疫，但与抗体产生相关基因和记忆细胞分化相关基因表达下调或没有显著变化，其表达模式的调控机制有待进一步研究。犬瘟热严重威胁大熊猫的种群安全，目前没有有效的免疫保护方法，因此，深入开展疫苗响应调控机制的研究、发展有效的免疫保护和靶向治疗尤为迫切。