2,4-二氯苯酚处理人肝细胞HL-7702转录组表达研究

发布时间：2018-11-22 20:19

【摘要】：氯酚类化合物作为一类持久性有机污染物,在医药、农药、工业生产和合成材料等多个行业广泛使用,不可避免会对生态环境造成难以估量的影响。氯酚类化合物在环境中分布较广,大气、水体及土壤中均有其踪影,而且它们通常难以降解,不仅危害到生态环境中各种动植物的正常生长和繁殖,甚至还可以通过食物链或直接接触,威胁人类健康。2,4-二氯苯酚(2,4-DCP)是氯酚类化合物中十分重要的一类物质,主要用工农业生产中于2,4-二氯苯氧乙酸、药物硫双二氯酚以及除草醚的合成。因其在生产活动中的大量使用,难以避免会被释放到环境中,从而会对生物体产生危害作用,2,4-DCP已被我国和美国等众多国家列入了优先控制污染物的名单。基于Illumina/Solexa高通量测序平台的转录组测序和分析技术(RNA-seq)可以在任何物种间实现单核苷酸水平的整体转录情况检测,通过检测结果可以进行转录本结构的分析及基因表达水平的分析。同时,还可以通过RNA-seq发现未知转录本和稀有转录本,全面的提供转录组信息。相比与传统的基因芯片技术,RNA-seq并不需要预先针对已知序列设计探针,也不受有无参考序列的限制。通过使用RNA-seq对转录组的研究,可以从整体水平上揭示基因结构以及基因功能,发现疾病发生过程或特定生物学过程中的分子机制。现有对于2,4-DCP的研究,多集中于环境中2,4-DCP的降解。2,4-DCP毒性机理的研究多处于生理指标的测定,而结合转录组测序技术的2,4-DCP毒性研究尚未见报道。本研究通过建立2,4-DCP处理人肝细胞HL-7702的转录组数据库,将转录组数据进行序列比对、功能注释及通路分析。通过对比2,4-DCP处理组细胞和对照组(无2,4-DCP处理)细胞的转录组差异表达,筛选出与2,4-DCP毒性等相关的基因,从而系统探讨其毒性机制。本论文得到的研究结果如下：1.通过RNA-seq技术,本研究共得到原始数据7.77 GB,其中对照组和2,4-DCP处理组分别得到54711542和51353236条的高质量配对短序列,其中能够比对到参考序列上的总reads条数,空白组有45318071条,2,4-DCP处理组有42090330条。覆盖度为90%-100%的基因分别占总基因数的56%和55%。2.通过对两组细胞转录组测序的结果进行差异基因表达分析,共筛选出1 521个差异表达基因(FDR≤0.001 AND |log2Ratio|≥1),其中上调基因数为795个,下调基因数为726个。3.经过GO功能显著性富集分析和KEGG通路富集分析,这些差异表达的基因主要参与固醇类的合成、细胞代谢、组织发育和内质网应激等生物过程。4.采用实时定量PCR技术(RT-qPCR)对ATP8A1、ATP8B4、CYP1A1、 CYP1B1、CYP2E1、CYP4F12、UGT1A6、UGT1A7、DHCR7和DHCR24等10个受2,4-DCP影响显著的基因进行了深入研究,结果表明RT-qPCR法与转录组分析结果一致。这些基因参与细胞膜结构的维持(ATP8A1和ATP8B4)、介导2,4-DCP在细胞内的代谢(CYP1A1、CYP1B1、CYP2E1、CYP4F12、UGT1A6和UGT1A7)并调控固醇类物质的合成(DHCR7和DHCR24),在细胞受2,4-DCP处理后,表达明显改变,也证明转录组结果反映了细胞内基因的真实表达情况。通过以上研究,本文发现2,4-DCP处理细胞后,其发挥毒性作用的可能机制有以下三点：(1)通过改变膜的磷脂结构进而影响细胞膜结构的稳定性。(2)通过代谢产生更具毒性的代谢中间产物或终产物,影响细胞内的正常生理功能。(3)通过影响固醇类激素的合成发挥其内分泌干扰的作用。总之,本文通过转录组测序从差异基因表达、GO功能富集分析和Pathway分析等方面,结合RT-qPCR验证实验全面系统地阐明了在2,4-DCP的作用下人肝细胞HL-7702的转录本特点,为进一步深入研究2,4-DCP的分子毒性机制奠定了研究基础。
[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.
【学位授予单位】：兰州大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R114;Q78

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