【摘要】：氯酚類化合物作為一類持久性有機(jī)污染物,在醫(yī)藥、農(nóng)藥、工業(yè)生產(chǎn)和合成材料等多個(gè)行業(yè)廣泛使用,不可避免會(huì)對(duì)生態(tài)環(huán)境造成難以估量的影響。氯酚類化合物在環(huán)境中分布較廣,大氣、水體及土壤中均有其蹤影,而且它們通常難以降解,不僅危害到生態(tài)環(huán)境中各種動(dòng)植物的正常生長(zhǎng)和繁殖,甚至還可以通過(guò)食物鏈或直接接觸,威脅人類健康。2,4-二氯苯酚(2,4-DCP)是氯酚類化合物中十分重要的一類物質(zhì),主要用工農(nóng)業(yè)生產(chǎn)中于2,4-二氯苯氧乙酸、藥物硫雙二氯酚以及除草醚的合成。因其在生產(chǎn)活動(dòng)中的大量使用,難以避免會(huì)被釋放到環(huán)境中,從而會(huì)對(duì)生物體產(chǎn)生危害作用,2,4-DCP已被我國(guó)和美國(guó)等眾多國(guó)家列入了優(yōu)先控制污染物的名單。基于Illumina/Solexa高通量測(cè)序平臺(tái)的轉(zhuǎn)錄組測(cè)序和分析技術(shù)(RNA-seq)可以在任何物種間實(shí)現(xiàn)單核苷酸水平的整體轉(zhuǎn)錄情況檢測(cè),通過(guò)檢測(cè)結(jié)果可以進(jìn)行轉(zhuǎn)錄本結(jié)構(gòu)的分析及基因表達(dá)水平的分析。同時(shí),還可以通過(guò)RNA-seq發(fā)現(xiàn)未知轉(zhuǎn)錄本和稀有轉(zhuǎn)錄本,全面的提供轉(zhuǎn)錄組信息。相比與傳統(tǒng)的基因芯片技術(shù),RNA-seq并不需要預(yù)先針對(duì)已知序列設(shè)計(jì)探針,也不受有無(wú)參考序列的限制。通過(guò)使用RNA-seq對(duì)轉(zhuǎn)錄組的研究,可以從整體水平上揭示基因結(jié)構(gòu)以及基因功能,發(fā)現(xiàn)疾病發(fā)生過(guò)程或特定生物學(xué)過(guò)程中的分子機(jī)制�，F(xiàn)有對(duì)于2,4-DCP的研究,多集中于環(huán)境中2,4-DCP的降解。2,4-DCP毒性機(jī)理的研究多處于生理指標(biāo)的測(cè)定,而結(jié)合轉(zhuǎn)錄組測(cè)序技術(shù)的2,4-DCP毒性研究尚未見(jiàn)報(bào)道。本研究通過(guò)建立2,4-DCP處理人肝細(xì)胞HL-7702的轉(zhuǎn)錄組數(shù)據(jù)庫(kù),將轉(zhuǎn)錄組數(shù)據(jù)進(jìn)行序列比對(duì)、功能注釋及通路分析。通過(guò)對(duì)比2,4-DCP處理組細(xì)胞和對(duì)照組(無(wú)2,4-DCP處理)細(xì)胞的轉(zhuǎn)錄組差異表達(dá),篩選出與2,4-DCP毒性等相關(guān)的基因,從而系統(tǒng)探討其毒性機(jī)制。本論文得到的研究結(jié)果如下：1.通過(guò)RNA-seq技術(shù),本研究共得到原始數(shù)據(jù)7.77 GB,其中對(duì)照組和2,4-DCP處理組分別得到54711542和51353236條的高質(zhì)量配對(duì)短序列,其中能夠比對(duì)到參考序列上的總reads條數(shù),空白組有45318071條,2,4-DCP處理組有42090330條。覆蓋度為90%-100%的基因分別占總基因數(shù)的56%和55%。2.通過(guò)對(duì)兩組細(xì)胞轉(zhuǎn)錄組測(cè)序的結(jié)果進(jìn)行差異基因表達(dá)分析,共篩選出1 521個(gè)差異表達(dá)基因(FDR≤0.001 AND |log2Ratio|≥1),其中上調(diào)基因數(shù)為795個(gè),下調(diào)基因數(shù)為726個(gè)。3.經(jīng)過(guò)GO功能顯著性富集分析和KEGG通路富集分析,這些差異表達(dá)的基因主要參與固醇類的合成、細(xì)胞代謝、組織發(fā)育和內(nèi)質(zhì)網(wǎng)應(yīng)激等生物過(guò)程。4.采用實(shí)時(shí)定量PCR技術(shù)(RT-qPCR)對(duì)ATP8A1、ATP8B4、CYP1A1、 CYP1B1、CYP2E1、CYP4F12、UGT1A6、UGT1A7、DHCR7和DHCR24等10個(gè)受2,4-DCP影響顯著的基因進(jìn)行了深入研究,結(jié)果表明RT-qPCR法與轉(zhuǎn)錄組分析結(jié)果一致。這些基因參與細(xì)胞膜結(jié)構(gòu)的維持(ATP8A1和ATP8B4)、介導(dǎo)2,4-DCP在細(xì)胞內(nèi)的代謝(CYP1A1、CYP1B1、CYP2E1、CYP4F12、UGT1A6和UGT1A7)并調(diào)控固醇類物質(zhì)的合成(DHCR7和DHCR24),在細(xì)胞受2,4-DCP處理后,表達(dá)明顯改變,也證明轉(zhuǎn)錄組結(jié)果反映了細(xì)胞內(nèi)基因的真實(shí)表達(dá)情況。通過(guò)以上研究,本文發(fā)現(xiàn)2,4-DCP處理細(xì)胞后,其發(fā)揮毒性作用的可能機(jī)制有以下三點(diǎn)：(1)通過(guò)改變膜的磷脂結(jié)構(gòu)進(jìn)而影響細(xì)胞膜結(jié)構(gòu)的穩(wěn)定性。(2)通過(guò)代謝產(chǎn)生更具毒性的代謝中間產(chǎn)物或終產(chǎn)物,影響細(xì)胞內(nèi)的正常生理功能。(3)通過(guò)影響固醇類激素的合成發(fā)揮其內(nèi)分泌干擾的作用。總之,本文通過(guò)轉(zhuǎn)錄組測(cè)序從差異基因表達(dá)、GO功能富集分析和Pathway分析等方面,結(jié)合RT-qPCR驗(yàn)證實(shí)驗(yàn)全面系統(tǒng)地闡明了在2,4-DCP的作用下人肝細(xì)胞HL-7702的轉(zhuǎn)錄本特點(diǎn),為進(jìn)一步深入研究2,4-DCP的分子毒性機(jī)制奠定了研究基礎(chǔ)。
[Abstract]:As a class of persistent organic pollutants, chlorophenols are widely used in many industries, such as medicine, pesticide, industrial production and synthetic materials, and it is inevitable to have an immeasurable effect on the ecological environment. The chlorinated phenolic compounds are widely distributed in the environment, and are found in the atmosphere, the water body and the soil, and are often difficult to degrade, not only can endanger the normal growth and propagation of various plants and plants in the ecological environment, but also can be directly contacted with the food chain or the direct contact, Threat to human health. 2,4-Dichlorophenol (2,4-DCP) is a very important kind of substance in the chlorophenol compound, which is mainly used for the synthesis of 2,4-dichlorophenoxyacetic acid, drug-sulfur double-dichlorophenol and phenyl ether in industrial and agricultural production. As a result of its extensive use in production activities, it is difficult to avoid being released into the environment, which will have a detrimental effect on the organism, and 2,4-DCP has been included in the list of priority control pollutants in a number of countries such as our country and the United States. The transcriptome sequencing and analysis technology (RNA-seq) based on the high-throughput sequencing platform of Illumina/ Solexa can be used to detect the whole transcription of the single-core acid level among any species, and the analysis of the structure and the analysis of the gene expression level can be carried out by the detection result. At the same time, an unknown transcript and a rare transcript can also be found by RNA-seq to provide a complete set of transcription-group information. Compared with the traditional gene chip technology, the RNA-seq does not need to design a probe in advance for a known sequence, and is not limited by the presence or absence of a reference sequence. By using the RNA-seq to study the transcription group, the gene structure and the gene function can be revealed from the whole level, and the molecular mechanism in the course of the disease or the specific biological process can be found. The 2, 4-DCP degradation of 2,4-DCP in the environment is more and more concentrated in the research of 2,4-DCP, and the toxicity of 2,4-DCP in combination with the transcriptome sequencing technology has not been reported. In this study, the transcriptome database of human hepatocytes HL-7702 was treated with 2,4-DCP, and the transcriptome data were compared, functional and path analysis. By comparing the differential expression of the transcription groups of the 2, 4-DCP treatment group cells and the control group (no 2,4-DCP treatment), genes related to the toxicity of 2,4-DCP and the like were selected, and the toxicity mechanism of the system was discussed. The results of the study are as follows: 1. The high-quality paired short-sequence of 54711542 and 51353236 in the control group and the 2, 4-DCP treatment group were obtained by RNA-seq technique. The number of total readth on the reference sequence can be compared with that of the control group. The blank group has 45318071, and the 2, 4-DCP treatment group has 42090330. The 90%-100% of the genes account for 56% and 55% of the total number of genes, respectively. A total of 1,521 differentially expressed genes (FDR-0. 001 AND | log2Rutio | -1) were screened by the differential gene expression analysis of the two groups of cell transcriptome sequencing. The number of up-regulated genes was 795 and the number of down-regulated genes was 726. The gene mainly involved in the synthesis of sterol, cell metabolism, tissue development and endoplasmic reticulum stress. A real-time quantitative PCR (RT-qPCR) was used to study the effects of 2, 4-DCP, such as CYP1A1, CYP8B4, CYP1A1, CYP1B1, CYP2E1, CYP4F12, UGT1A6, UGT1A7, DHCR7 and DHCR24, and the results showed that the RT-qPCR method was consistent with the analysis of the transcriptome. These genes are involved in the maintenance of cell membrane structures (CYP8A1 and B8B4), and mediate the metabolism of 2,4-DCP in the cell (CYP1A1, CYP1B1, CYP2E1, CYP4F12, UGT1A6 and UGT1A7) and regulate the synthesis of sterol substances (DHCR7 and DHCR24), and the expression is obviously changed after the cells are treated with 2,4-DCP. It is also shown that the results of the transcription group reflect the true expression of the gene in the cell. Through the above research, we find that the possible mechanism of the toxicity of 2,4-DCP has the following three points: (1) by changing the phospholipid structure of the membrane, the stability of the cell membrane structure is also affected. (2) a more toxic metabolic intermediate product or end product is produced by metabolism, affecting normal physiological functions within the cell. and (3) play the role of endocrine disturbance by influencing the synthesis of the sterol-like hormone. In conclusion, the transcription of human hepatocytes HL-7702 under the action of 2,4-DCP in the presence of 2,4-DCP was described in a systematic and systematic way through the transcriptome sequencing from the aspects of differential gene expression, GO function enrichment analysis and pathway analysis. In order to further study the molecular toxicity mechanism of 2,4-DCP, the research foundation was established.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R114;Q78

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