三丁基錫肝細胞毒性的轉錄組學研究
發(fā)布時間：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錫化合物,可對海洋生物和哺乳動物造成嚴重的危害,主要包括生殖毒性、神經毒性、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胞形態(tài)層面,而在基因和蛋白質水平展開的研究非常少,因此TBT誘導肝毒性的分子機制仍不清楚。本研究選用人正常來源的肝細胞HL7702,運用毒物基因組學技術對TBT影響肝細胞的基因表達水平進行研究。方法:1.用MTT法檢測不同濃度的TBT(0、2、4、6、8、10μM)暴露2 h后對細胞增殖的影響。2.HL7702細胞暴露于TBT后提取總RNA,并用Nano Drop核酸檢測儀和1%甲醛變性瓊脂糖凝膠電泳檢測RNA質量。3.Affymetrix表達譜芯片分析TBT對全基因組轉錄水平的影響。4.用SAM軟件篩選對照組和TBT暴露組之間的差異表達基因,要求FDR5%且實驗組基因表達水平/對照組基因表達水平2。5.用GO數(shù)據(jù)庫進行基因注釋和生物學解釋,用DAVID數(shù)據(jù)庫進行基因功能聚類和信號通路分析。6.芯片研究中發(fā)現(xiàn)的13個凋亡相關差異表達基因進行實時熒光定量PCR(qRT-PCR)驗證。7.通過流式細胞術,用Annexin V-FITC細胞凋亡檢測試劑盒分析HL7702細胞暴露于不同濃度的TBT 2 h后的細胞凋亡率。8.用IPA軟件進行上游信號分析、下游信號分析,構建TBT誘導細胞凋亡的信號通路圖。結果:1.根據(jù)MTT結果,選取0、2、6、10μM的TBT染毒開展后續(xù)實驗,并把2、6、10μM分別作為低濃度、中濃度和高濃度。2.根據(jù)芯片實驗,細胞暴露于低濃度、中濃度和高濃度的TBT后分別出現(xiàn)770,1028,1221個差異表達基因。3.信號通路分析結果表明,細胞凋亡是TBT肝毒性的主要原因。4.對芯片篩選得到的13個凋亡相關基因用q RT-PCR進行驗證,變化趨勢與芯片結果一致。5.流式細胞術表明,低濃度的TBT暴露后細胞的凋亡率并沒有發(fā)生改變,但是中、高濃度的TBT暴露后,凋亡細胞數(shù)量明顯增加,并呈現(xiàn)劑量-效應關系。6.IPA軟件提示HSP家族、激酶和TNF受體等共同介導了TBT誘導的細胞凋亡。7.HL7702細胞暴露于TBT后,p53基因信號通路、整合素信號通路、微絲細胞骨架調控也在通路分析時被富集,說明這些信號通路也可能參與了TBT誘導的肝毒性。結論:全基因組表達譜芯片顯示誘導細胞凋亡是TBT肝毒性作用的主要原因,死亡受體途徑、線粒體途徑和內質網(wǎng)途徑全部參與了TBT誘導的細胞凋亡。
[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.
【學位授予單位】：寧波大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：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.
【學位授予單位】：寧波大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R114
【相似文獻】
相關期刊論文 前10條
1 加貝;應用電子捕獲檢測器氣相色譜及質譜測定魚體中的三丁基錫[J];國外醫(yī)學(衛(wèi)生學分冊);1989年01期
2 周群芳,江桂斌,劉稷燕;三丁基錫化合物對稀有泩鯽的急慢性毒理研究[J];中國科學(B輯 化學);2003年02期
3 朱欣,應李強,徐進,徐立紅;三丁基錫對正常人胚胎羊膜細胞凋亡的誘導作用[J];環(huán)境科學學報;2005年09期
4 黃周英,王重剛,左正宏,趙揚,陳奕欣;三丁基錫對文蛤鰓的抗氧化酶活性及脂質過氧化的影響[J];環(huán)境科學學報;2005年10期
5 曾懷才;賀慶芝;陳鋒;;氯化三丁基錫致巨噬細胞凋亡作用[J];中國公共衛(wèi)生;2010年01期
6 張亞莉;孫玉濤;翁登坡;徐立紅;;三丁基錫對小鼠脾、肝和腦組織蛋白磷酸酶2A活性的影響[J];癌變·畸變·突變;2013年03期
7 朱欣;邢鳴鸞;樓建林;徐立紅;;三丁基錫對正常人胚胎羊膜細胞凋亡相關蛋白表達的影響[J];環(huán)境科學學報;2006年11期
8 李娟;萬益群;申明月;秦峰;;氣相色譜-火焰光度法測定自來水中三丁基錫和三苯基錫[J];食品科學;2009年22期
9 張亞莉;徐立紅;;三丁基錫產生細胞毒性效應的分子機制研究[J];癌變·畸變·突變;2013年04期
10 葉懷莊,邢鳴鸞,錢江,俞智勇,徐立紅;三丁基錫對小鼠臟器組織總氧自由基清除能力的影響[J];環(huán)境與健康雜志;2005年03期
相關會議論文 前10條
1 曾懷才;;氯化三丁基錫對巨噬細胞的凋亡作用[A];湖南省預防醫(yī)學會勞動衛(wèi)生專業(yè)委員會2008年學術交流會議論文集[C];2008年
2 陳海剛;馬勝偉;張U，
本文編號：2394986
本文鏈接：http://sikaile.net/yixuelunwen/yufangyixuelunwen/2394986.html
最近更新
教材專著
