【作者】 赵静；

【导师】 王全凯；

【作者基本信息】 吉林农业大学 ， 预防兽医学， 2016， 博士

【摘要】 子宫内膜炎是一种重要产科疾病,严重影响着奶牛业经济效益。生殖道微生物群落对奶牛子宫内膜炎的发生起关键作用,研究其作用机制不仅为开发防治奶牛子宫内膜炎的微生态制剂提供参考,而且也能为阐明该病的发病机理奠定基础。本研究利用PCR-DGGE和荧光定量PCR等技术比较研究了产后不同阶段健康和子宫内膜炎奶牛子宫菌群结构的演替规律,利用RNA-seq技术分析了LPS(细菌脂多糖)对奶牛子宫内膜细胞全基因组表达谱的影响,以期解析奶牛子宫内膜炎相关微生态菌群及其与宿主的相互作用机制。采集健康和子宫内膜炎奶牛产后7d、14d、25d、36d和46d子宫分泌物样品。首先利用PCR-DGGE技术比较研究产后不同阶段健康和子宫内膜炎奶牛子宫细菌多样性。结果表明,健康和子宫内膜炎奶牛产后子宫内细菌多样性均呈现先升高后降低的演替规律。产后7d,羊创伤球菌、坏死梭杆菌和微小单胞菌仅存在于患病组,而解脲支原体、甲烷炎菌属和产气荚膜梭菌在两组奶牛中均存在。产后第14d,解脲支原体和羊创伤球菌仅存在于健康组,而卟啉单胞菌、解肝素拟杆菌、甲烷炎菌属和大肠杆菌在健康组和患病组均存在。产后25d,患病组细菌多样性高于健康组,卟啉单胞菌、门多萨假单胞菌和甲烷炎菌属在两组均存在。产后36d,患病组细菌多样性显著高于健康组,卟啉单胞菌、埃希氏菌、放线杆菌、甲烷炎菌属和坏死梭杆菌等仅存在于患病组。产后46d,健康和患病组子宫内细菌多样性显著降低,且两组间差异不显著。基于PCR-DGGE结果,筛选出6种特异性的菌属,应用荧光定量PCR检测其在健康和患病组产后不同阶段数量变化规律。结果显示:各菌属数量随产后不同阶段呈现动态变化,且产后46d各菌数量在健康组和患病组趋于一致。解脲支原体在6种菌属中数量最高,随时间呈下降趋势,且患病组均高于健康组。坏死梭杆菌数量在健康和患病奶牛产后均呈现先增加后减少的变化规律,且产后7d到36d期间,患病组坏死梭杆菌数量均高于健康组。产后不同阶段门多萨假单胞菌和甲烷炎菌的数量在健康和子宫内膜炎奶牛间均差异不显著。产后25d,患病组卟啉单胞菌数量显著高于健康组。羊创伤球菌数量呈现先升高后降低的变化,且仅在产后14d和25d患病组数量显著高于健康组。结合PCR-DGGE结果,坏死梭杆菌和卟啉单胞菌可能是奶牛子宫内膜炎的主要致病菌。RNA-seq结果表明,LPS处理奶牛子宫内膜炎细胞24h后,有108个差异基因。GO分类统计及富集分析结果显示,GO分类统计中差异基因注释到生物过程的GO term共24个,注释到细胞组分的GO term共15个,注释到分子功能的GO term共10个。GO富集分析中差异基因富集到生物过程的GO term共68个,富集到细胞组分的GO term共4个,富集到分子功能的GO term共8个。KEGG通路富集分析结果显示,共富集102个信号通路,其中20个信号通路显著富集,其中与炎症相关的差异富集通路包括TNF、NOD-like receptor和NF-kappa B等,从高通量结果中选择4个关键基因,利用实时荧光定量PCR验证其表达量,验证结果证明了高通量测序技术的可靠性。本研究不仅利用分子生物学技术研究了产后不同阶段健康和子宫内膜炎奶牛子宫菌群结构的变化规律,明确了二者间的差异,而且利用RNA-seq等技术获得了LPS处理奶牛子宫内膜细胞的全基因组表达谱,研究结果不仅为揭示奶牛子宫内膜炎发病机制奠定基础,而且也为开发新型治疗药物提供了参考。更多还原

【Abstract】 Endometritis is a common obstetrical disease, which leads to huge economic losses in dairy industry. Reproductive tracts microecology plays an important role in the occurrence of cow endometritis, therefore, exploring its mechanism not only can lay theoretical basis for developing probiotics used for the prevention and treatment of cow endometritis, but also provide reference for clarifying pathogenesis of endometritis. In the present study, in order to clarify cow endometritis associated microbiota and its interaction mechanisms with the host, change of bacterial diversity in reproductive tracts of healthy cows and endometritis cows were investigated using PCR-DGGE and Real-time PCR, then, RNA-seq technique was used to analyse genomic expression profiling of cow endometrial cellsUterine samples were collected from healthy cows and endometritis cows on days 7, 14, 25, 36, and 46 postpartum and then PCR-DGGE was used to investigate their microbial community structure. The results showed that uterine bacterial diversity in healthy and endometritis groups followed the same succession rule: increasing firstly then decreasing. On day 7, Helcococcus ovis, Fusobacterium necrophorum and Parvimonas micra were only detected in endometritis group, while Ureaplasma diversum, Parasporobacterium paucivorans, Methanotorris igneus and Clostridium perfringens were detected in both groups. On day 14, Ureaplasma diversum and Helcococcus ovis were only detected in healthy group, while Porphyromonas levii, Bacteroides heparinolyticus, Methanotorris igneus and Escherichia coli were detected in both groups. On day 25, Porphyromonas levii, Methanotorris igneus and Pseudomonas mendocina were detected in both groups. On day 36, bacterial diversity in endometritis group was significantly higher than healthy group, and Porphyromonas levii, Micrococcus luteus, Escherichia fergusonii, Actinobacillus seminis, Methanotorris igneus and Fusobacterium necrophorum were only detected in endometritis group. On day 46, bacterial diversity in both groups decreased sinificantly and there was no significant difference between the two groups.Based on results of PCR-DGGE, number of six bacteria in uterus of healthy and endometritis cows postpartum was investigated using Real-time PCR. The results showed that number of the bacteria changed dynamically as different stage of postpartum and the number was almost the same in healthy and endometritis cows on day 46. Number of Ureaplasma diversum was the highest among the bacteria and deacreased as the time expanded, furthermore, the number in endometritis group was higher than healthy group. Number of Fusobacterium necrophorum in both groups increased firstly then decreased, which was higher in endometritis group than healthy group from day 7 to day 36. There was no significant difference on number of Methanotorris igneus and Pseudomonas mendocina between healthy and endometritis groups during different stages of postpartum. On day 25, number of Porphyromonas levii in endometritis group was significantly higher than healthy group. Number of Helcococcus ovis increased firstly then decreased, and it was higher in endometrtis group than healthy group on day 14 and 25. Combined with results of PCR-DGGE, Fusobacterium necrophorum and Porphyromonas levii may be the main pathogenic bacteria of cow endometritis.Results of RNA-seq showed that 108 genes were differentially expressed inlcuding 90 up-regulated genes and 18 down-regulated genes in cow endometrial cells of LPS treatment group compared to control group. Results of GO classification and enrichment of 108 different genes showed that, 24 GO terms were annotated to the biological process, 15 GO terms were annotated to the cell component, and 10 GO terms were annotated to the molecular function. 68 GO terms were enrichmented to the biological process, 4 GO terms were enrichmented to the cell component and 8 GO terms were enrichmented to the molecular function.The KEGG results revealed that 20 signaling pathway were more highly expressed in LPS treated groups including TNF signaling pathway, NOD-like receptor signaling pathway and NF-kappa B signaling pathway which are closely related to inflammation process. Then four genes were selected to verify the results of RNA-seq using Real-time PCR and the results showed that RNA-seq conducted in this study was accurate.In conclusion, the present study demonstrated change of bacterial diversity in uterus of healhty cows and endometritis cows and their difference using PCR-DGGE and Real-time PCR, Futhermore, genomic expression profiling of cow endometrial cells was analysized using RNA-seq technique. The results not only lay a foundation for calrify pathogenesis of endometritis, but also provided reference for developing probiotics to prevent and treat cow endometritis.更多还原