基于高通量测序技术探讨生物信息学在氟化物毒理机制和水环境微生物耐药机制研究中的应用
发布时间：2018-09-03 18:53
【摘要】：[目的]运用RNA-Seq转录组测序技术和生物信息学相关理论知识,定量计算组织中氟暴露动物生殖系统中信号通路和基因表达丰度,描绘氟中毒小鼠的睾丸组织中整体的基因表达谱,旨在阐明氟的生殖毒性在睾丸组织中的表现,进一步揭示氟对雄性动物生殖系统及精子代谢障碍的分子基础。[方法]选取8周龄性成熟雄性小鼠60只,随机平均分成三组,分别为对照组和50mg/L NaF组、100mg/L NaF组,自由饮水摄氟56天(小鼠精子发生周期40±2天),取每组5只小鼠睾丸组织,提取总RNA (Trizol),采用第二代高通量测序平台Illumina Hiseq2000进行barcoding转录组测序(RNA-seq)。产生的序列数据进行tophat和Cufflinks比对和分析得到差异的基因后，使用Ingenuity Pathway软件构建由差异表达基因组成的通路。寻找靶基因和相关通路,利用qRT-PCR进行基因验证。[结果]与对照组相比,50mg/L NaF组和100mg/L NaF组分别有120个和198个基因差异表达,同时分别有19条和33条通路具有显著差异性。从功能上讲，这些通路主要与细胞免疫应答,神经传递,生物合成代谢途径,细胞生长、增殖及发育,细胞应激反应与凋亡等作用相关。1.50mg/L NaF组的差异通路主要与氧化应激和细胞凋亡相关，，而100 mg/L NaF组的信号通路中与细胞免疫功能相关的路径表现明显,其中有4条通路表达显著并参与白细胞介素17(IL-17)的调节。选取参与其路径调节的基因IL-17A, IL-17RA, IL-17RC, MAP2K3, MAP2K6, PIK3R1, MAPKAPK2, MAP2K1 and MAP2K2进行定量分析得到,100 mg/L NaF组中IL-17RA, IL-17RC, MAP2K1, MAP2K2, MAP2K3和MAPKAPK2显著表达,与测序结果一致。2.50mg/L NaF和100 mg/L NaF两个处理组中,细胞间粘附连接信号转导通路都表达显著，因此对100 mg/L NaF和对照组进行第二次测序验证得到,氟对小鼠睾丸作用后,对小鼠机体中的102条通路都产生了不同程度的影响,其中,最具有显著差异性的通路主要与免疫系统有关系,同时检测到上皮细胞附着连接通路显著表达。筛选得到的通路中有4条通路与血睾屏障间细胞的紧密连接作用相关,对其相关基因MAGI1, ARPC5, SSX2IP, PVRL2, DNM3和CLDN14转录组表达水平定量验证，ARPC5和CLDN14表达显著下降,SSX2IP和PVRL2表达显著上升,与测序结果一致。[结论]氟对小鼠睾丸造成的损伤主要表现在三个方面：1.氟暴露首先引起小鼠睾丸组织的氧化应激与细胞凋亡反应。2.氟对机体的免疫系统引起的影响：IL-17信号通路的高表达是免疫系统对高氟入侵睾丸组织后的应答;PI13激酶、丝裂素活化蛋白激酶(MAPK)以及TGF-β家族中的细胞因子在维持免疫豁免和生精过程起到了重要的调控作用。3.氟对小鼠睾丸中细胞间紧密连接作用的影响正是氟对小鼠血睾屏障产生损伤的一个重要体现。高浓度的氟暴露可以引起相关基因蛋白的表达异常,使得细胞膜、膜蛋白的结构和功能异常,最终影响血睾屏障间的紧密连接的开放和闭合。在这种情况下,精子就会受到自身免疫系统的攻击,使得生精过程紊乱。所有的结果(代谢途径和典型的基因)为进一步深入研究氟中毒的分子机制提供新的思路和线索,包括生殖与发育、免疫反应、氧化应激和细胞调控机制等方面的研究。[目的]利用第二代高通量测序技术研究沿海地区水产养殖区域水环境徽生物不同种类抗生素耐药基因(包括已知的和未知的)的分布规律与流行特征,探寻药物残留与相应耐药基因分布流行的关联性。[方法]采集15个中国沿海水产养殖区域的表层沉积物,提取样品的中DNA,构建测序文库,采用第二代高通量测序平台Illumina Hiseq2000进行宏基因组测序,原始序列经过过滤与比对后,进行水环境中耐药流行特征的综合分析：一方面分析水环境中微生物的种类和丰度,分析微生物群体特征;一方面建立水环境中耐药基因库,分析耐药基因的数量、种类和分布情况。[结果]通过分析水环境中抗菌药物耐药性基因库中微生物耐药基因的种类和数量,研究结果显示：1.15个地区共检测到322种耐药基因,其中包括耐氨基糖苷类基因60种,β内酰胺类耐药基因60种,对大环内酯类、林可酰胺类和链阳霉素(MLSB)抗菌药物的多重耐药基因45种,多重耐药基因44种,四环素类耐药基因23种,多肽类耐药基因16种,耐氯霉素类基因13种,喹诺酮类耐药基因13种,磺胺类耐药基因12种。相比去其他的耐药基因类型,氨基糖苷类耐药基因和β内酰胺类耐药基因呈多地区普遍流行性。2.51.55%,31.37%和12.42%的耐药基因是分布由于酶活性的破坏,外排泵的作用以及细胞靶细胞的改变产生的细菌耐药。与之对应的,主要由于酶破坏引起的耐药抗生素主要有氨基糖苷类和β内酰胺类,磺胺类的耐药主要由于药物经外排泵排出细胞外引起。喹诺酮类主要是改变或保护药物作用的靶位引起。3.耐药基因的分布有明显的海域地理分布特征：以大连和莱阳为代表的渤海区域主要呈多重耐药和喹诺酮耐药流行;黄海海域主要是氨基糖苷类耐药明显;中国东海海域主要的耐药基因类型是耐氨基糖苷类、耐喹诺酮类以及多重耐药,特别是杭州湾、象山港和温州耐药基因污染严重;从宁德到三亚的中国南海沿海主要呈多重耐药趋势。[结论]该研究结果对揭示耐药基因在环境中的存在和分布具有重要意义，对环境中兽药安全标准评价系统的确立具有指导性,为有效控制动物和人类耐药性的产生,保障食品安全和人类健康提供了有力信息和依据。同时,第二代高通量测序技术的应用极大地提高了我们从宏观与整体的水平认知环境微生物耐药基因多样性,灵敏地探测出环境因子随外界环境的改变而发生的极其微弱的变化,对于我们研究微生物与环境的关系、环境与人类的健康有着重要的理论和现实意义。
[Abstract]:[Objective] Using RNA-Seq transcriptome sequencing technology and bioinformatics related knowledge, we quantitatively calculated the signal pathways and gene expression abundance in the reproductive system of fluoride-exposed animals, and described the overall gene expression profile in the testicular tissues of fluorosis mice, in order to clarify the reproductive toxicity of fluoride in the testicular tissues, and further uncover the expression profile. [Methods] Sixty 8-week-old male mice were randomly divided into three groups: control group and 50 mg/L NaF group, 100 mg/L NaF group, free drinking water for 56 days (mouse spermatogenesis cycle 40 Izol, using the second-generation high-throughput sequencing platform Illumina Hiseq2000 for barcoding transcriptome sequencing (RNA-seq). Sequence data generated by tophat and Cufflinks alignment and analysis of the different genes, using Ingenuity Pathway software to construct the differential expression of genes composed of the pathway. [Results] Compared with the control group, 120 and 198 genes were differentially expressed in 50 mg/L NaF group and 100 mg/L NaF group respectively, and 19 and 33 pathways were significantly different. 1.50 mg/L NaF group was mainly associated with oxidative stress and apoptosis, while 100 mg/L NaF group was significantly associated with cellular immune function. Four of these pathways were significantly expressed and involved in the regulation of interleukin-17 (IL-17). Quantitative analysis of the regulated genes IL-17A, IL-17RA, IL-17RC, MAP2K3, MAP2K6, PIK3R1, MAPKAPK2, MAP2K1 and MAP2K2 showed that IL-17RA, IL-17RC, MAP2K1, MAP2K2, MAP2K3 and MAPKAPK2 were significantly expressed in the 100 mg/L NaF group. The results were consistent with the sequencing results. 2.50 mg/L NaF and 100 mg/L NaF treatment groups, and the intercellular adhesion junction signal transduction was observed. Therefore, the second sequencing of 100 mg/L NaF and control group showed that fluoride had different effects on 102 pathways in mice after testicular action. The most significant difference was mainly related to the immune system and epithelial cell adhesion junction was detected. Quantitative analysis of the transcriptome expression levels of MAGI1, ARPC5, SSX2IP, PVRL2, DNM3 and CLDN14 showed that the expression of ARPC5 and CLDN14 decreased significantly, while the expression of SSX2IP and PVRL2 increased significantly, consistent with the sequencing results. Fluoride exposure first caused oxidative stress and apoptosis in mouse testicular tissue. 2. Fluoride-induced effects on the immune system: the high expression of IL-17 signaling pathway is the response of the immune system to high fluoride invasion of testicular tissue; PI13 kinase, mitogen activation Protein kinase (MAPK) and cytokines in the TGF-beta family play important roles in maintaining immune immune immunity and spermatogenesis. 3. The effect of fluoride on tight junction between cells in mouse testis is an important manifestation of the damage of blood-testicular barrier caused by fluoride exposure in high concentration. In this case, the sperm will be attacked by the autoimmune system, resulting in the disorder of spermatogenesis. All the results (metabolic pathways and typical genes) will provide further insight into fluorosis. Molecular mechanisms provide new ideas and clues, including reproduction and development, immune response, oxidative stress and cellular regulation mechanisms. [Objective] To study the distribution of antibiotic resistance genes (including known and unknown) in different species of aquatic microorganisms in coastal aquaculture areas using second-generation high-throughput sequencing techniques. [Methods] The surface sediments from 15 coastal aquaculture areas in China were collected to extract the DNA from the samples and construct a sequencing library. The second generation high throughput sequencing platform Illumina Hiseq2000 was used to sequence the macrogenome. After comparison, the epidemiological characteristics of drug resistance in water environment were analyzed comprehensively. On the one hand, the species and abundance of microorganisms in water environment were analyzed, and the characteristics of microbial population were analyzed. The results showed that 322 resistance genes were detected in 1.15 regions, including 60 aminoglycoside-resistant genes, 60 beta-lactam-resistant genes, 45 multi-resistance genes to macrolides, lincolamides and streptomycin (MLSB), and 44 multi-resistance genes. There are 23 tetracycline resistance genes, 16 polypeptide resistance genes, 13 chloramphenicol resistance genes, 13 quinolone resistance genes, and 12 sulfonamide resistance genes. Genes are distributed as a result of the destruction of enzyme activity, efflux pump action and changes in cell target cells resulting in bacterial resistance. Correspondingly, drug-resistant antibiotics mainly due to enzyme damage are aminoglycosides and beta-lactams. Sulfonamides resistance is mainly due to the drug efflux pump discharged from the cell. 3. Distribution of drug-resistant genes has obvious geographical characteristics in the sea: the Bohai Sea region represented by Dalian and Laiyang is mainly multidrug-resistant and quinolone-resistant epidemic; the Yellow Sea is mainly aminoglycoside-resistant; and the East China Sea is the main type of drug-resistant genes. Aminoglycosides, quinolones resistance and multiple drug resistance, especially in Hangzhou Bay, Xiangshan Harbor and Wenzhou, are seriously contaminated by drug resistance genes. The main trend of multi-drug resistance is from Ningde to Sanya along the coast of South China Sea. The establishment of the quasi-evaluation system is instructive and provides powerful information and basis for effectively controlling the emergence of drug resistance in animals and humans, ensuring food safety and human health. At the same time, the application of the second generation high-throughput sequencing technology has greatly improved our understanding of the environmental microbial resistance gene diversity and sensitivity from the macro and overall level. It is of great theoretical and practical significance for us to study the relationship between microorganism and environment and between environment and human health to detect the extremely weak changes of environmental factors with the changes of external environment.
【学位授予单位】：山西农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：X171.5;Q811.4
本文编号：2220867
[Abstract]:[Objective] Using RNA-Seq transcriptome sequencing technology and bioinformatics related knowledge, we quantitatively calculated the signal pathways and gene expression abundance in the reproductive system of fluoride-exposed animals, and described the overall gene expression profile in the testicular tissues of fluorosis mice, in order to clarify the reproductive toxicity of fluoride in the testicular tissues, and further uncover the expression profile. [Methods] Sixty 8-week-old male mice were randomly divided into three groups: control group and 50 mg/L NaF group, 100 mg/L NaF group, free drinking water for 56 days (mouse spermatogenesis cycle 40 Izol, using the second-generation high-throughput sequencing platform Illumina Hiseq2000 for barcoding transcriptome sequencing (RNA-seq). Sequence data generated by tophat and Cufflinks alignment and analysis of the different genes, using Ingenuity Pathway software to construct the differential expression of genes composed of the pathway. [Results] Compared with the control group, 120 and 198 genes were differentially expressed in 50 mg/L NaF group and 100 mg/L NaF group respectively, and 19 and 33 pathways were significantly different. 1.50 mg/L NaF group was mainly associated with oxidative stress and apoptosis, while 100 mg/L NaF group was significantly associated with cellular immune function. Four of these pathways were significantly expressed and involved in the regulation of interleukin-17 (IL-17). Quantitative analysis of the regulated genes IL-17A, IL-17RA, IL-17RC, MAP2K3, MAP2K6, PIK3R1, MAPKAPK2, MAP2K1 and MAP2K2 showed that IL-17RA, IL-17RC, MAP2K1, MAP2K2, MAP2K3 and MAPKAPK2 were significantly expressed in the 100 mg/L NaF group. The results were consistent with the sequencing results. 2.50 mg/L NaF and 100 mg/L NaF treatment groups, and the intercellular adhesion junction signal transduction was observed. Therefore, the second sequencing of 100 mg/L NaF and control group showed that fluoride had different effects on 102 pathways in mice after testicular action. The most significant difference was mainly related to the immune system and epithelial cell adhesion junction was detected. Quantitative analysis of the transcriptome expression levels of MAGI1, ARPC5, SSX2IP, PVRL2, DNM3 and CLDN14 showed that the expression of ARPC5 and CLDN14 decreased significantly, while the expression of SSX2IP and PVRL2 increased significantly, consistent with the sequencing results. Fluoride exposure first caused oxidative stress and apoptosis in mouse testicular tissue. 2. Fluoride-induced effects on the immune system: the high expression of IL-17 signaling pathway is the response of the immune system to high fluoride invasion of testicular tissue; PI13 kinase, mitogen activation Protein kinase (MAPK) and cytokines in the TGF-beta family play important roles in maintaining immune immune immunity and spermatogenesis. 3. The effect of fluoride on tight junction between cells in mouse testis is an important manifestation of the damage of blood-testicular barrier caused by fluoride exposure in high concentration. In this case, the sperm will be attacked by the autoimmune system, resulting in the disorder of spermatogenesis. All the results (metabolic pathways and typical genes) will provide further insight into fluorosis. Molecular mechanisms provide new ideas and clues, including reproduction and development, immune response, oxidative stress and cellular regulation mechanisms. [Objective] To study the distribution of antibiotic resistance genes (including known and unknown) in different species of aquatic microorganisms in coastal aquaculture areas using second-generation high-throughput sequencing techniques. [Methods] The surface sediments from 15 coastal aquaculture areas in China were collected to extract the DNA from the samples and construct a sequencing library. The second generation high throughput sequencing platform Illumina Hiseq2000 was used to sequence the macrogenome. After comparison, the epidemiological characteristics of drug resistance in water environment were analyzed comprehensively. On the one hand, the species and abundance of microorganisms in water environment were analyzed, and the characteristics of microbial population were analyzed. The results showed that 322 resistance genes were detected in 1.15 regions, including 60 aminoglycoside-resistant genes, 60 beta-lactam-resistant genes, 45 multi-resistance genes to macrolides, lincolamides and streptomycin (MLSB), and 44 multi-resistance genes. There are 23 tetracycline resistance genes, 16 polypeptide resistance genes, 13 chloramphenicol resistance genes, 13 quinolone resistance genes, and 12 sulfonamide resistance genes. Genes are distributed as a result of the destruction of enzyme activity, efflux pump action and changes in cell target cells resulting in bacterial resistance. Correspondingly, drug-resistant antibiotics mainly due to enzyme damage are aminoglycosides and beta-lactams. Sulfonamides resistance is mainly due to the drug efflux pump discharged from the cell. 3. Distribution of drug-resistant genes has obvious geographical characteristics in the sea: the Bohai Sea region represented by Dalian and Laiyang is mainly multidrug-resistant and quinolone-resistant epidemic; the Yellow Sea is mainly aminoglycoside-resistant; and the East China Sea is the main type of drug-resistant genes. Aminoglycosides, quinolones resistance and multiple drug resistance, especially in Hangzhou Bay, Xiangshan Harbor and Wenzhou, are seriously contaminated by drug resistance genes. The main trend of multi-drug resistance is from Ningde to Sanya along the coast of South China Sea. The establishment of the quasi-evaluation system is instructive and provides powerful information and basis for effectively controlling the emergence of drug resistance in animals and humans, ensuring food safety and human health. At the same time, the application of the second generation high-throughput sequencing technology has greatly improved our understanding of the environmental microbial resistance gene diversity and sensitivity from the macro and overall level. It is of great theoretical and practical significance for us to study the relationship between microorganism and environment and between environment and human health to detect the extremely weak changes of environmental factors with the changes of external environment.
【学位授予单位】：山西农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：X171.5;Q811.4
本文编号：2220867
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