三丁基锡肝细胞毒性的转录组学研究

发布时间：2018-12-29 15:17

【摘要】：目的:三丁基锡(tributyltin,TBT)是一种广泛使用的有机锡化合物,可对海洋生物和哺乳动物造成严重的危害,主要包括生殖毒性、神经毒性、免疫毒性以及肝毒性等。肝脏作为外源化合物代谢最主要的器官,也是TBT攻击的主要靶器官之一。TBT现有的研究主要停留在细胞形态层面,而在基因和蛋白质水平展开的研究非常少,因此TBT诱导肝毒性的分子机制仍不清楚。本研究选用人正常来源的肝细胞HL7702,运用毒物基因组学技术对TBT影响肝细胞的基因表达水平进行研究。方法:1.用MTT法检测不同浓度的TBT(0、2、4、6、8、10μM)暴露2 h后对细胞增殖的影响。2.HL7702细胞暴露于TBT后提取总RNA,并用Nano Drop核酸检测仪和1%甲醛变性琼脂糖凝胶电泳检测RNA质量。3.Affymetrix表达谱芯片分析TBT对全基因组转录水平的影响。4.用SAM软件筛选对照组和TBT暴露组之间的差异表达基因,要求FDR5%且实验组基因表达水平/对照组基因表达水平2。5.用GO数据库进行基因注释和生物学解释,用DAVID数据库进行基因功能聚类和信号通路分析。6.芯片研究中发现的13个凋亡相关差异表达基因进行实时荧光定量PCR(qRT-PCR)验证。7.通过流式细胞术,用Annexin V-FITC细胞凋亡检测试剂盒分析HL7702细胞暴露于不同浓度的TBT 2 h后的细胞凋亡率。8.用IPA软件进行上游信号分析、下游信号分析,构建TBT诱导细胞凋亡的信号通路图。结果:1.根据MTT结果,选取0、2、6、10μM的TBT染毒开展后续实验,并把2、6、10μM分别作为低浓度、中浓度和高浓度。2.根据芯片实验,细胞暴露于低浓度、中浓度和高浓度的TBT后分别出现770,1028,1221个差异表达基因。3.信号通路分析结果表明,细胞凋亡是TBT肝毒性的主要原因。4.对芯片筛选得到的13个凋亡相关基因用q RT-PCR进行验证,变化趋势与芯片结果一致。5.流式细胞术表明,低浓度的TBT暴露后细胞的凋亡率并没有发生改变,但是中、高浓度的TBT暴露后,凋亡细胞数量明显增加,并呈现剂量-效应关系。6.IPA软件提示HSP家族、激酶和TNF受体等共同介导了TBT诱导的细胞凋亡。7.HL7702细胞暴露于TBT后,p53基因信号通路、整合素信号通路、微丝细胞骨架调控也在通路分析时被富集,说明这些信号通路也可能参与了TBT诱导的肝毒性。结论:全基因组表达谱芯片显示诱导细胞凋亡是TBT肝毒性作用的主要原因,死亡受体途径、线粒体途径和内质网途径全部参与了TBT诱导的细胞凋亡。
[Abstract]:Objective: tin triDing Ji (tributyltin,TBT) is a widely used organotin compound, which can cause serious harm to marine life and mammals, including reproductive toxicity, neurotoxicity, immune toxicity and hepatotoxicity. As the most important organ of exogenous compound metabolism, liver is also one of the main target organs of TBT attack. The existing studies of TBT mainly focus on the cellular morphology, but very little on the gene and protein levels. Therefore, the molecular mechanism of TBT induced hepatotoxicity remains unclear. In this study, HL7702, from normal human hepatocytes was used to study the effect of TBT on gene expression in hepatocytes. Methods: 1. MTT assay was used to detect the effect of different concentrations of TBT (0 02O2O4O4C6O6ON8U 10 渭 M) on cell proliferation 2 hours after exposure to TBT. Total RNA, was extracted from 2.HL7702 cells after TBT exposure. Nano Drop nucleic acid detector and 1% formaldehyde denatured agarose gel electrophoresis were used to detect the quality of RNA. 3.Affymetrix expression microarray was used to analyze the effect of TBT on the whole genome transcription level. 4. SAM software was used to screen the differentially expressed genes between the control group and the TBT exposed group. The FDR5% and the gene expression level of the experimental group and the control group were required to be 2.5 / 2.5 respectively. GO database was used for gene annotation and biological interpretation, and DAVID database was used for gene functional clustering and signal pathway analysis. Thirteen differentially expressed apoptosis-related genes were identified by real-time fluorescent quantitative PCR (qRT-PCR). The apoptosis rate of HL7702 cells exposed to different concentrations of TBT for 2 h was analyzed by flow cytometry with Annexin V-FITC cell apoptosis assay kit. The upstream signal analysis and downstream signal analysis were performed with IPA software to construct the signal pathway diagram of TBT induced apoptosis. Results: 1. According to the results of MTT, 10 渭 M TBT was selected for the follow-up experiment, and the 10 渭 M of 20 渭 M was used as the low concentration, the middle concentration and the high concentration of 2. 2. According to microarray experiment, 770 ~ 1028 ~ 1221 differentially expressed genes were found in cells exposed to low, medium and high concentrations of TBT, respectively. Signal pathway analysis showed that apoptosis was the main cause of TBT hepatotoxicity. 4. The 13 apoptosis-related genes screened by microarray were verified by Q RT-PCR. Flow cytometry showed that there was no change in apoptosis rate after exposure to low concentration of TBT, but the number of apoptotic cells increased significantly after exposure to moderate and high concentration of TBT, and showed a dose-effect relationship. 6.IPA software showed that HSP family. The apoptosis induced by TBT was mediated by kinase and TNF receptor. After 7.HL7702 cells were exposed to TBT, p53 gene signaling pathway, integrin signal pathway and microfilament cytoskeleton regulation were also enriched when the pathway was analyzed. These signaling pathways may also be involved in TBT induced hepatotoxicity. Conclusion: the whole genome expression microarray showed that apoptosis was the main cause of TBT hepatotoxicity. The death receptor pathway, mitochondrial pathway and endoplasmic reticulum pathway were all involved in the apoptosis induced by TBT.
【学位授予单位】：宁波大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114

【相似文献】