【摘要】：[目的]運(yùn)用RNA-Seq轉(zhuǎn)錄組測(cè)序技術(shù)和生物信息學(xué)相關(guān)理論知識(shí),定量計(jì)算組織中氟暴露動(dòng)物生殖系統(tǒng)中信號(hào)通路和基因表達(dá)豐度,描繪氟中毒小鼠的睪丸組織中整體的基因表達(dá)譜,旨在闡明氟的生殖毒性在睪丸組織中的表現(xiàn),進(jìn)一步揭示氟對(duì)雄性動(dòng)物生殖系統(tǒng)及精子代謝障礙的分子基礎(chǔ)。[方法]選取8周齡性成熟雄性小鼠60只,隨機(jī)平均分成三組,分別為對(duì)照組和50mg/L NaF組、100mg/L NaF組,自由飲水?dāng)z氟56天(小鼠精子發(fā)生周期40±2天),取每組5只小鼠睪丸組織,提取總RNA (Trizol),采用第二代高通量測(cè)序平臺(tái)Illumina Hiseq2000進(jìn)行barcoding轉(zhuǎn)錄組測(cè)序(RNA-seq)。產(chǎn)生的序列數(shù)據(jù)進(jìn)行tophat和Cufflinks比對(duì)和分析得到差異的基因后，使用Ingenuity Pathway軟件構(gòu)建由差異表達(dá)基因組成的通路。尋找靶基因和相關(guān)通路,利用qRT-PCR進(jìn)行基因驗(yàn)證。[結(jié)果]與對(duì)照組相比,50mg/L NaF組和100mg/L NaF組分別有120個(gè)和198個(gè)基因差異表達(dá),同時(shí)分別有19條和33條通路具有顯著差異性。從功能上講，這些通路主要與細(xì)胞免疫應(yīng)答,神經(jīng)傳遞,生物合成代謝途徑,細(xì)胞生長(zhǎng)、增殖及發(fā)育,細(xì)胞應(yīng)激反應(yīng)與凋亡等作用相關(guān)。1.50mg/L NaF組的差異通路主要與氧化應(yīng)激和細(xì)胞凋亡相關(guān)，，而100 mg/L NaF組的信號(hào)通路中與細(xì)胞免疫功能相關(guān)的路徑表現(xiàn)明顯,其中有4條通路表達(dá)顯著并參與白細(xì)胞介素17(IL-17)的調(diào)節(jié)。選取參與其路徑調(diào)節(jié)的基因IL-17A, IL-17RA, IL-17RC, MAP2K3, MAP2K6, PIK3R1, MAPKAPK2, MAP2K1 and MAP2K2進(jìn)行定量分析得到,100 mg/L NaF組中IL-17RA, IL-17RC, MAP2K1, MAP2K2, MAP2K3和MAPKAPK2顯著表達(dá),與測(cè)序結(jié)果一致。2.50mg/L NaF和100 mg/L NaF兩個(gè)處理組中,細(xì)胞間粘附連接信號(hào)轉(zhuǎn)導(dǎo)通路都表達(dá)顯著，因此對(duì)100 mg/L NaF和對(duì)照組進(jìn)行第二次測(cè)序驗(yàn)證得到,氟對(duì)小鼠睪丸作用后,對(duì)小鼠機(jī)體中的102條通路都產(chǎn)生了不同程度的影響,其中,最具有顯著差異性的通路主要與免疫系統(tǒng)有關(guān)系,同時(shí)檢測(cè)到上皮細(xì)胞附著連接通路顯著表達(dá)。篩選得到的通路中有4條通路與血睪屏障間細(xì)胞的緊密連接作用相關(guān),對(duì)其相關(guān)基因MAGI1, ARPC5, SSX2IP, PVRL2, DNM3和CLDN14轉(zhuǎn)錄組表達(dá)水平定量驗(yàn)證，ARPC5和CLDN14表達(dá)顯著下降,SSX2IP和PVRL2表達(dá)顯著上升,與測(cè)序結(jié)果一致。[結(jié)論]氟對(duì)小鼠睪丸造成的損傷主要表現(xiàn)在三個(gè)方面：1.氟暴露首先引起小鼠睪丸組織的氧化應(yīng)激與細(xì)胞凋亡反應(yīng)。2.氟對(duì)機(jī)體的免疫系統(tǒng)引起的影響：IL-17信號(hào)通路的高表達(dá)是免疫系統(tǒng)對(duì)高氟入侵睪丸組織后的應(yīng)答;PI13激酶、絲裂素活化蛋白激酶(MAPK)以及TGF-β家族中的細(xì)胞因子在維持免疫豁免和生精過(guò)程起到了重要的調(diào)控作用。3.氟對(duì)小鼠睪丸中細(xì)胞間緊密連接作用的影響正是氟對(duì)小鼠血睪屏障產(chǎn)生損傷的一個(gè)重要體現(xiàn)。高濃度的氟暴露可以引起相關(guān)基因蛋白的表達(dá)異常,使得細(xì)胞膜、膜蛋白的結(jié)構(gòu)和功能異常,最終影響血睪屏障間的緊密連接的開(kāi)放和閉合。在這種情況下,精子就會(huì)受到自身免疫系統(tǒng)的攻擊,使得生精過(guò)程紊亂。所有的結(jié)果(代謝途徑和典型的基因)為進(jìn)一步深入研究氟中毒的分子機(jī)制提供新的思路和線(xiàn)索,包括生殖與發(fā)育、免疫反應(yīng)、氧化應(yīng)激和細(xì)胞調(diào)控機(jī)制等方面的研究。[目的]利用第二代高通量測(cè)序技術(shù)研究沿海地區(qū)水產(chǎn)養(yǎng)殖區(qū)域水環(huán)境徽生物不同種類(lèi)抗生素耐藥基因(包括已知的和未知的)的分布規(guī)律與流行特征,探尋藥物殘留與相應(yīng)耐藥基因分布流行的關(guān)聯(lián)性。[方法]采集15個(gè)中國(guó)沿海水產(chǎn)養(yǎng)殖區(qū)域的表層沉積物,提取樣品的中DNA,構(gòu)建測(cè)序文庫(kù),采用第二代高通量測(cè)序平臺(tái)Illumina Hiseq2000進(jìn)行宏基因組測(cè)序,原始序列經(jīng)過(guò)過(guò)濾與比對(duì)后,進(jìn)行水環(huán)境中耐藥流行特征的綜合分析：一方面分析水環(huán)境中微生物的種類(lèi)和豐度,分析微生物群體特征;一方面建立水環(huán)境中耐藥基因庫(kù),分析耐藥基因的數(shù)量、種類(lèi)和分布情況。[結(jié)果]通過(guò)分析水環(huán)境中抗菌藥物耐藥性基因庫(kù)中微生物耐藥基因的種類(lèi)和數(shù)量,研究結(jié)果顯示：1.15個(gè)地區(qū)共檢測(cè)到322種耐藥基因,其中包括耐氨基糖苷類(lèi)基因60種,β內(nèi)酰胺類(lèi)耐藥基因60種,對(duì)大環(huán)內(nèi)酯類(lèi)、林可酰胺類(lèi)和鏈陽(yáng)霉素(MLSB)抗菌藥物的多重耐藥基因45種,多重耐藥基因44種,四環(huán)素類(lèi)耐藥基因23種,多肽類(lèi)耐藥基因16種,耐氯霉素類(lèi)基因13種,喹諾酮類(lèi)耐藥基因13種,磺胺類(lèi)耐藥基因12種。相比去其他的耐藥基因類(lèi)型,氨基糖苷類(lèi)耐藥基因和β內(nèi)酰胺類(lèi)耐藥基因呈多地區(qū)普遍流行性。2.51.55%,31.37%和12.42%的耐藥基因是分布由于酶活性的破壞,外排泵的作用以及細(xì)胞靶細(xì)胞的改變產(chǎn)生的細(xì)菌耐藥。與之對(duì)應(yīng)的,主要由于酶破壞引起的耐藥抗生素主要有氨基糖苷類(lèi)和β內(nèi)酰胺類(lèi),磺胺類(lèi)的耐藥主要由于藥物經(jīng)外排泵排出細(xì)胞外引起。喹諾酮類(lèi)主要是改變或保護(hù)藥物作用的靶位引起。3.耐藥基因的分布有明顯的海域地理分布特征：以大連和萊陽(yáng)為代表的渤海區(qū)域主要呈多重耐藥和喹諾酮耐藥流行;黃海海域主要是氨基糖苷類(lèi)耐藥明顯;中國(guó)東海海域主要的耐藥基因類(lèi)型是耐氨基糖苷類(lèi)、耐喹諾酮類(lèi)以及多重耐藥,特別是杭州灣、象山港和溫州耐藥基因污染嚴(yán)重;從寧德到三亞的中國(guó)南海沿海主要呈多重耐藥趨勢(shì)。[結(jié)論]該研究結(jié)果對(duì)揭示耐藥基因在環(huán)境中的存在和分布具有重要意義，對(duì)環(huán)境中獸藥安全標(biāo)準(zhǔn)評(píng)價(jià)系統(tǒng)的確立具有指導(dǎo)性,為有效控制動(dòng)物和人類(lèi)耐藥性的產(chǎn)生,保障食品安全和人類(lèi)健康提供了有力信息和依據(jù)。同時(shí),第二代高通量測(cè)序技術(shù)的應(yīng)用極大地提高了我們從宏觀與整體的水平認(rèn)知環(huán)境微生物耐藥基因多樣性,靈敏地探測(cè)出環(huán)境因子隨外界環(huán)境的改變而發(fā)生的極其微弱的變化,對(duì)于我們研究微生物與環(huán)境的關(guān)系、環(huán)境與人類(lèi)的健康有著重要的理論和現(xiàn)實(shí)意義。
[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.
【學(xué)位授予單位】：山西農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：X171.5;Q811.4


本文編號(hào)：2220867