三丁基錫肝細(xì)胞毒性的轉(zhuǎn)錄組學(xué)研究
發(fā)布時(shí)間：2018-12-29 15:17
【摘要】：目的:三丁基錫(tributyltin,TBT)是一種廣泛使用的有機(jī)錫化合物,可對(duì)海洋生物和哺乳動(dòng)物造成嚴(yán)重的危害,主要包括生殖毒性、神經(jīng)毒性、免疫毒性以及肝毒性等。肝臟作為外源化合物代謝最主要的器官,也是TBT攻擊的主要靶器官之一。TBT現(xiàn)有的研究主要停留在細(xì)胞形態(tài)層面,而在基因和蛋白質(zhì)水平展開(kāi)的研究非常少,因此TBT誘導(dǎo)肝毒性的分子機(jī)制仍不清楚。本研究選用人正常來(lái)源的肝細(xì)胞HL7702,運(yùn)用毒物基因組學(xué)技術(shù)對(duì)TBT影響肝細(xì)胞的基因表達(dá)水平進(jìn)行研究。方法:1.用MTT法檢測(cè)不同濃度的TBT(0、2、4、6、8、10μM)暴露2 h后對(duì)細(xì)胞增殖的影響。2.HL7702細(xì)胞暴露于TBT后提取總RNA,并用Nano Drop核酸檢測(cè)儀和1%甲醛變性瓊脂糖凝膠電泳檢測(cè)RNA質(zhì)量。3.Affymetrix表達(dá)譜芯片分析TBT對(duì)全基因組轉(zhuǎn)錄水平的影響。4.用SAM軟件篩選對(duì)照組和TBT暴露組之間的差異表達(dá)基因,要求FDR5%且實(shí)驗(yàn)組基因表達(dá)水平/對(duì)照組基因表達(dá)水平2。5.用GO數(shù)據(jù)庫(kù)進(jìn)行基因注釋和生物學(xué)解釋,用DAVID數(shù)據(jù)庫(kù)進(jìn)行基因功能聚類和信號(hào)通路分析。6.芯片研究中發(fā)現(xiàn)的13個(gè)凋亡相關(guān)差異表達(dá)基因進(jìn)行實(shí)時(shí)熒光定量PCR(qRT-PCR)驗(yàn)證。7.通過(guò)流式細(xì)胞術(shù),用Annexin V-FITC細(xì)胞凋亡檢測(cè)試劑盒分析HL7702細(xì)胞暴露于不同濃度的TBT 2 h后的細(xì)胞凋亡率。8.用IPA軟件進(jìn)行上游信號(hào)分析、下游信號(hào)分析,構(gòu)建TBT誘導(dǎo)細(xì)胞凋亡的信號(hào)通路圖。結(jié)果:1.根據(jù)MTT結(jié)果,選取0、2、6、10μM的TBT染毒開(kāi)展后續(xù)實(shí)驗(yàn),并把2、6、10μM分別作為低濃度、中濃度和高濃度。2.根據(jù)芯片實(shí)驗(yàn),細(xì)胞暴露于低濃度、中濃度和高濃度的TBT后分別出現(xiàn)770,1028,1221個(gè)差異表達(dá)基因。3.信號(hào)通路分析結(jié)果表明,細(xì)胞凋亡是TBT肝毒性的主要原因。4.對(duì)芯片篩選得到的13個(gè)凋亡相關(guān)基因用q RT-PCR進(jìn)行驗(yàn)證,變化趨勢(shì)與芯片結(jié)果一致。5.流式細(xì)胞術(shù)表明,低濃度的TBT暴露后細(xì)胞的凋亡率并沒(méi)有發(fā)生改變,但是中、高濃度的TBT暴露后,凋亡細(xì)胞數(shù)量明顯增加,并呈現(xiàn)劑量-效應(yīng)關(guān)系。6.IPA軟件提示HSP家族、激酶和TNF受體等共同介導(dǎo)了TBT誘導(dǎo)的細(xì)胞凋亡。7.HL7702細(xì)胞暴露于TBT后,p53基因信號(hào)通路、整合素信號(hào)通路、微絲細(xì)胞骨架調(diào)控也在通路分析時(shí)被富集,說(shuō)明這些信號(hào)通路也可能參與了TBT誘導(dǎo)的肝毒性。結(jié)論:全基因組表達(dá)譜芯片顯示誘導(dǎo)細(xì)胞凋亡是TBT肝毒性作用的主要原因,死亡受體途徑、線粒體途徑和內(nèi)質(zhì)網(wǎng)途徑全部參與了TBT誘導(dǎo)的細(xì)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.
【學(xué)位授予單位】：寧波大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R114
[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.
【學(xué)位授予單位】：寧波大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R114
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