【作者】 左之才；

【导师】 崔恒敏；

【作者基本信息】 四川农业大学 ， 临床兽医学， 2012， 博士

【摘要】 目的：PP在高密度饲养的现代大型规模化养猪场所造成的损失及危害相当严重。PP的病原学、流行特点及检测方法等一直是国内外研究人员和学者关注的焦点；关于APP感染对猪机体组织损伤及免疫相关的系统研究较为少见。本研究旨在：(1)建立PP人工感染疾病模型；(2)监测感染APP后,猪的病症、血液常规及生化指标、抗氧化功能、免疫状况(红细胞免疫、T细胞免疫与体液免疫及细胞因子水平)及病理变化；(3)利用含43,603个探针的Agilent猪类全基因组芯片检测感染APP猪的肺和肺门淋巴结基因表达谱变化,探索感染组织损伤的分子机制。结果：1.采用鼻腔气雾法,按猪每千克体重鼻腔喷雾含APP3.5-4×107CFU/ml的APP(I型)稀释液0.25ml,成功建立了PP疾病模型。2.感染猪的WBC、GRA及MON的数量及比例显著增高(P<0.01),LYM的数量及比例显著降低(P<0.01)。血清GLB含量显著增高(0.01<P<0.05),ALB含量、A/G显著降低(0.01<P<0.05)；AST活力极显著升高(P<0.01)、LT/ST显著减小(0.01<P<0.05)；TBil含量、IBil含量及IB/DB极显著增高(P<0.01)；CRE含量、BUN含量略有升高；GLU浓度、AKP活力、Ca含量、Ca/P极显著下降(P<0.01),P含量显著上升(0.01<P<0.05)3.感染猪的RBC-C3bRR极显著升高(P<0.01),RBC-ICR降低；外周血CD3+T细胞降低、CD3+CD4+T细胞极显著降低(P<0.01)、CD3+CD8+T细胞和CD4+/CD8+显著降低(0.01<P<0.05)；SI显著减小(0.01<P<0.05)。血清IgA、IgE含量升高,IgG含量降低。血清SAA、CRP浓度略有升高；TGF-13含量显著减少(0.01<P<0.05),IL-1、IL-2、IL-6含量减少；PDGF、IFN-γ、TNF-a含量升高。4.感染猪的肺组织除CAT活力显著增高(0.01<P<0.05)外,GSH-PX、SOD活力均极显著降低(P<0.01)；MDA含量显著降低(0.01<P<0.05),-OH含量极显著降低(P<0.01)；T-AOC极显著降低(P<0.01)。肺门淋巴结CAT和GSH-PX活力极显著增高(P<0.01)；-OH含量极显著降低(P<0.01)；T-AOC极显著下降(P<0.01)。5.猪类全基因组芯片表达谱的结果与分析：(1)芯片扫描样点均匀规则,信号强度高,信噪比高,探针特异性强,芯片检出率高,集中在53%-74%区间。(2)用T检验方法分析得到感染与非感染APP猪肺及肺门淋巴结的差异表达基因及其交集。(3)SOM分析将差异表达基因聚为9类,涉及到免疫增强、Toll-like受体信号通路、磷脂酰肌醇信号系统通路,粘着连接通路等过程。样本HCL将相同组织聚为一类,即肺组织和肺门淋巴结样本各自聚类；同组织(肺或淋巴)感染与非感染样本各自聚类。(4)PCA分析显示：差异基因在同组织以疾病动物高于健康动物为主要表达模式；在同一个体以肺门淋巴结高于肺组织为主要表达模式。(5)基于GO功能分类的GCT结果显示：在肺组织,基因变异程度达到显著和极显著(PErmineJ<0.05(?)口0.01)的GO分类89个,与免疫有关的27个。在肺门淋巴结,基因变异程度达显著和极显著(PErmineJ<0.05和0.01)的GO分类有272个,与免疫有关的50个。(6)GSEA结果显示：基因在与感染有关的,如Fcepsilon RI信号传导途径、NOD-like受体信号通路、急性骨髓性白血病、癌症、孕酮介导卵母细胞成熟、剪接体、第Ⅱ型糖尿病、类固醇激素生物合成等pathway中显著富集,并得知这些pathway发挥调控作用的方向性(上调或下调)。Leading edge analysis发现多个调控机体抗感染与组织损伤修复可能具有重要或潜在作用的基因,在不同的pathway中多次出现。(7)基因芯片的可靠性评估结果显示：基因芯片内的变异系数为2.57%-5.50%。qRT-PCR验证结果显示,两种不同实验方法检测结果为正相关,相关系数平均为0.861±0.127。结论：1.利用Agilent猪类全基因组芯片研究感染APP猪肺脏及肺门淋巴结的表达谱,筛选出了对猪免疫、抗感染及组织损伤可能有重大影响和重要调控作用的基因31个以及有较大研究价值的pathway19个,初步揭示了APP感染对猪肺及肺门淋巴结损伤的分子机理。2.感染APP猪的细胞免疫和体液免疫功能下降,红细胞免疫功能增强；总抗氧化能力下降。更多还原

【Abstract】 Objectives:Porcine pleuropeumonia has caused very serious loss and damage in the modern high-stocking density large-scale breeding farms. The etiology, epidemic characteristics, test methods and other research of porcine pleuropneumonia has been a focus of attention of the various countries’researchers and scholars; while the systematic studies of tissue damage caused by bacteria infection and immune-related indexes on the body of pig are rare. Therefore, this study aimed at:(1) Establishing the pleuropneumoniae pathological model of artificial infection;(2) Observating and studying the swine symptoms, blood indicators and biochemical parameters, antioxidant indicators, immune parameters (red blood cell immunity, T cell-mediated immunity, humoral immunity and cytokine levels) and pathological changes post-infection with APP;(3) Using the Agilent swine genome-wide microarray containing43,603probes to detect the changes in gene expression of infected pigs’lungs and hilar nodes, to explore the molecular mechanisms of injury infection.Results:1. The porcine pleuropneumonia disease model were builded succesfully by nasal spraying0.25ml diluent contained3.5-4×107CFU/ml APP every kilogram weight.2. The number of leukocytes, and the numbers and the percentages of granulocyte and monocytes were all increased significantly (P<0.01) while the number and the percentage of lymphocytes were decreased significantly (P<0.01). The content of GLB in infected-APP pigs serum was increased significantly (0.01<P<0.05), and ALB, A/G were decreased significantly (0.01<P<0.05); the activity of AST were increased (P<0.01) while LT/ST was decreased (0.01<P<0.05); the contents of TBil, IBil and IB/DB were increased extremely significantly (P<0.01); the contents of CRE and BUN were both increased slightly; the concentration of GLU, the activity of AKP, the content of Ca and Ca/P were decreased significantly (P<0.01), the content of P was increased significantly (0.01P<0.05)3. The ratios of RBC-C3b rosette were increased significantly (P<0.01) and the ratios of IC rosette were decreased (P<0.01); the percentage of CD3+T cells in the peripheral blood and CD3+CD4+T cells were decreased extremely significantly (P<0.01) while the percentage of CD3+CD8+T cells and CD4+/CD8+were decreased significantly (0.01<P<0.05); the SI was reduced evidently(0.01<P<0.05); the contents of IgA and IgE were increased while the count of IgG was decreased. The counts of serum SAA and c-reactive protein were increased slightly; The count of TGF-β was decreased significantly (0.01<P<0.05), and IL-1, IL-2and IL-6were decreased; The counts of PDGF, IFN-y and TNF-a were increased.4. In the lung tissues of infected-APP pig, SOD and GSH-PX activities were decreased (P<0.01), and CAT activities were increased (0.01<P<0.05); MDA and-OH contents were decreased (0.01<P<0.05or P<0.01); T-AOC were decreased (P<0.01). In the HLN of infected-APP pig, CAT and GSH-PX activities were were increased (P<0.01);-OH count and T-AOC contents were decreased (P<0.01).5. The main results of pig genome-wide expression analysis of the chip are as follows:(1)Pig-chip scan results were showed that the sampling point was uniform and inerratic, the signal intensity was high, the signal to noise ratio was high, the specificity of probe was strong, detection rate of chip was high, and it concentrated in the range53%～74%.(2) T-tests were used to detect the differentially expression with a pig’s lung and HLN in infected and non-infected:having got the DE genes and these intersection in the infected and non-infected lung and HLN.(3) The SOM of DE genes revealed that the genes were classified into9kinds, included the processes of immune enhancement, Toll-like receptor signaling pathway, phosphatidylinositol signaling pathway, and adhesion connecting pathway. The HCL of DE genes revealed that lung samples and HLN samples each clustered together; infected and non-infected samples of the same tissue each clustered together.(4) PCA revealed that the main expression pattern in the same kind of tissue was that DE genes in disease animals were higher than those in healthy ones; while in the same individual, DE genes in lymphoid tissue were higher than those in lung tissue.(5) GCT based on GO analysis revealed that89GO terms that the degree of genetic variation reached significant and highly significant (PErmineJ<0.05and0.01) were found in lung, including27GO terms related with immune.272GO terms that the degree of genetic variation reached significant and highly significant (PErmineJ<0.05and0.01) were found in HLN, including50GO terms related with immune.(6) GSEA revealed that genes were enriched significantly in a number of infection-related pathway including Fc epsilon RI (one of IgE receptors) signaling transduction pathway, NOD-like receptor signaling pathway, acute myeloid leukemia, cancer, progesterone-mediated oocyte maturation, spliceosome, Ⅱ diabetes, steroid hormones biosynthesis, and have got the direction (upward or downward) of these pathway that plays a regulatory role in each group. By Leading edge analysis found several potential genes appeared in a different pathway in the high-frequency and may had an important role in the regulation of body resistance to infection and the repair of damaged tissue by Leading edge analysis found.(7) The reliability evaluation results showed that the largest relative Standard Deviation (RSD) was5.50%and the smallest RSD was only2.57%in the gene chip. QRT-PCR verification results showed that there were a positive correlation between two detection results from different experimental methods in lung and lymphoid tissue and correlation coefficient averaged at0.861±0.127.Conclusion and innovation1. Initially reveal the molecular basis of the APP infection damage to the lung and HLN by using the Agilent pigs genome-wide microarray expression profiling.31genes were selected which might have a significant impact and important role for the pigs’ immune, anti-infection and anti-tissue damage, as well as19pathways of greater research value.2. The cellular immune and humoral immune function of the infected-APP pigs decreased, while the erythrocyte immune function increased. The T-AOC of infected-APP pigs were decreased.更多还原