TIM-3/Caspase通路介导通城猪和大白猪感染PRRSV后胸腺细胞凋亡差异的分子机制

Porcine reproductive and respiratory syndrome virus (PRRSV) cause thymocyte apoptosis in pigs, which would affect T cell mediated immune response and lead to immunosuppression. We have found that Tongcheng pigs showed stronger cellular immune response compared to Large White Pigs when they were artificial infected with PRRSV. In addition, thymocyte apoptosis rate of Tongcheng pigs was lower than Large White pigs. We have found that TIM-3 gene may regulate thymocyte apoptosis based on our previous studies. To investigate molecular mechanisms of thymocyte apoptosis caused by TIM-3, first we will investigate the expression of TIM-3 and Caspase family genes in thymus and thymocytes collected from PRRSV infected Tongcheng pigs and Large White pigs. Then, functional analysis of TIM-3 will be conducted in primary thymocytes to investigate its roles involve in thymocyte apoptosis. To further clarify the mechanism of differently expression of TIM-3 between Tongcheng and Large White pigs, we will study transcriptional regulation and post-transcriptional regulation of TIM-3. In this study, the whole-genome resequencing data will be used to detect SNPs in two different breeds, and potential functional SNPs will be confirmed by different methods and measured that if they will affect transcriptional start. In the end, we will predict potential miRNA (miR-145-5p) that will affect expression of TIM-3 and verify relationship between miRNAs and TIM-3 by different methods. The project will have significant scientific and practical results, which can explain the reason that Tongcheng have stronger cellular immune response during PRRSV infection and provide candidate genes and variants for PRRS resistant breeding.

猪繁殖与呼吸综合征病毒（PRRSV）感染引起机体细胞免疫抑制。前期研究揭示胸腺细胞凋亡参与了通城猪与大白猪细胞免疫抑制的品种差异调控，并发现TIM-3是调控胸腺细胞凋亡的关键基因。所以，本项目拟以TIM-3基因为切入点，用通城猪与大白猪作对照，系统探讨TIM-3及其介导的Caspase通路在胸腺细胞凋亡调控中的角色。本项目拟开展以下研究：1)从mRNA和蛋白质水平阐明TIM-3及Caspase家族基因在胸腺和胸腺细胞中的表达定位;2)以胸腺细胞为材料，超表达和抑制表达TIM-3，通过Caspase通路活化与否以相关生化指标系统评价TIM-3的细胞凋亡调控作用;3)通过调控元件深入分析TIM-3基因的品种差异表达机制，并探讨品种间差异表达miR-145-5p对TIM-3的转录后调控作用。研究成果将有助于理解猪的抗猪繁殖与呼吸综合征（PRRS）分子机制，对猪PRRS抗病育种也具有重要意义。

猪繁殖与呼吸综合征（PRRS）俗称“猪蓝耳病”，是由猪繁殖与呼吸综合征病毒（PRRSV）引起的高度传染性疾病,严重危害我国养猪业。PRRSV感染导致机体免疫抑制，造成病毒持续性感染并引起其他病原的继发感染。前期研究发现对PRRS具有抗病性差异的通城猪和大白猪感染PRRSV后细胞免疫应答存在差异。胸腺细胞(T细胞前体)凋亡程度不同直接影响通城猪和大白猪细胞免疫应答差异。因此，鉴定影响胸腺细胞凋亡的关键基因，找到不同品种猪调控胸腺细胞凋亡以及细胞免疫应答差异的分子遗传机理对PRRS抗病育种具有重要意义。本研究通过人工感染PRRSV试验发现大白猪感染PRRSV后胸腺组织损伤程度和胸腺细胞凋亡程度均高于通城猪。胸腺细胞和淋巴结基因表达和蛋白表达分析发现通城猪和大白猪感染PRRSV后抗原呈递能力、细胞免疫应答和细胞凋亡程度存在差异。大白猪感染PRRSV后细胞凋亡通路被激活，促细胞凋亡相关基因显著上调。本研究发现控制淋巴细胞凋亡的关键基因TIM-3在大白猪胸腺细胞中显著上调表达，而在通城猪胸腺细胞中上调不明显。在293T细胞和PK-15细胞中超表达TIM-3显著上调Caspase-3蛋白表达，同时增加细胞凋亡程度。通过对TIM-3进行转录和转录后调控研究发现ssc-miR-145-5p通过转录后调控介导不同品种差异表达TIM-3。qRT-PCR发现通城猪胸腺细胞ssc-miR-145-5p在感染PRRSV后显著上调，而大白猪没有明显变化。随后本研究在293T细胞和PK-15细胞中证实ssc-miR-145-5p通过直接靶向3’UTR区域调控TIM-3表达和细胞凋亡。为鉴定与宿主细胞免疫相关的候选基因和分子标记，利用人工感染PRRSV的通城猪和大白猪高代横交群体进行全基因组SNP分型和外周血淋巴细胞百分比测定，通过关联分析鉴定出rs81257542(DDX17)、 rs334594334(BTG1)和rs80901155(CNTNAP5)三个影响细胞免疫应答的候选基因和分子标记，建立两种分子标记快速检测方法。在本项目资助下，发表研究论文6篇，申请专利1项，投稿会议论文3篇，培养3名研究生。综上所述，本研究获得成果将有助于解析通城猪细胞免疫强的抗病遗传机理，对PRRS抗病育种具有重要意义。