1,2:3,4-二環(huán)氧丁烷誘導細胞DNA雙鏈斷裂損傷及其修復機制研究
發(fā)布時間：2018-08-26 19:16
【摘要】：研究背景1,3-丁二烯(1,3-butadiene,BD)是一種重要的工業(yè)原料,用于合成橡膠、塑料等石油化工和制造行業(yè)。美國將BD列為前40位重要的化學品之一,應用范圍廣,職業(yè)接觸人群多。此外,汽車尾氣、香煙煙霧及食用油油煙中也發(fā)現(xiàn)BD普遍存在,屬于室內(nèi)具有慢性空氣危害的9種優(yōu)先污染物之一。因此BD不僅是一種職業(yè)暴露污染物,更是一種重要的環(huán)境空氣污染物,其危害范圍已從職業(yè)工人擴展到普通人群。BD可以誘導嚙齒動物多器官腫瘤的形成,人群研究也顯示出白血病發(fā)病相關的陽性結果,因而被IARC和EPA列為確定的人類致癌物(1組)。BD的致癌效應主要來自于體內(nèi)代謝產(chǎn)生的環(huán)氧化產(chǎn)物:1,2-環(huán)氧-3-丁烯(EB),1,2-二羥基-3,4-環(huán)氧丁烯(EBD)和1,2:3,4-二環(huán)氧丁烯(DEB)。上述環(huán)氧化產(chǎn)物均能與DNA形成加合物,造成遺傳物質的損傷,其中DEB的活性最強,因而目前對BD致癌機制研究多聚焦于闡明其遺傳損傷效應。既往研究發(fā)現(xiàn)BD及其代謝物可以誘導DNA雙鏈斷裂和染色體損傷,但是BD誘導DNA雙鏈斷裂損傷后的修復機制并不清楚。本課題組前期在人群研究中發(fā)現(xiàn)BD暴露與核質橋(NPB)比例升高相關。NPB是染色體損傷的標志和細胞癌變的重要環(huán)節(jié),研究BD誘導NPB形成的機制,將為闡明BD的致癌作用過程,預防和減少BD所誘導的染色體損傷和人群腫瘤的發(fā)生提供重要依據(jù)。有研究提示,NPB的形成可能與DNA雙鏈斷裂后,經(jīng)非同源末端連接(NHEJ)等錯誤修復形成雙著絲粒染色體,在細胞分裂后期分別牽引到兩個子細胞核有關。由此我們推測,BD及其代謝物誘導DNA雙鏈斷裂后,可能通過影響主要的DNA雙鏈斷裂修復通路,即HR和NHEJ修復通路,造成錯誤修復效率的提高,從而引起NPB形成。因此,本課題以BD活性代謝物DEB為對象,在明確其誘導人淋巴母細胞DNA雙鏈斷裂和染色體損傷的條件下,研究DEB對DNA雙鏈斷裂修復通路的影響,進一步驗證修復通路在DEB致NPB形成中的作用,為深入探討B(tài)D的遺傳損傷機制提供依據(jù)。研究內(nèi)容1.DEB的細胞毒性效應研究建立DEB染毒處理的人淋巴母細胞TK6細胞模型模型。分別采用MTS和Ed U方法檢測DEB對細胞活性和增殖能力的影響。采用流式細胞儀分析技術,分別以PI和Annix V-FITC/PI染色檢測DEB對細胞周期和細胞凋亡的影響。2.DEB誘導細胞遺傳損傷的檢測采用胞質分裂阻滯微核試驗(CBMNT)分析DEB對細胞染色體的損傷(微核率、核質橋率、核芽突率及核分裂指數(shù));采用Western blot和免疫熒光技術,檢測DNA雙鏈斷裂標志物(γ-H2AX)的表達情況。3.DEB對細胞DNA雙鏈斷裂損傷修復的影響采用含有HR修復效率檢測報告底物的MCF-7細胞,檢測DEB處理后HR修復效率的變化。采用Cell-free方法,提取細胞總蛋白,與32P標記的線性質粒共同作用,檢測DEB處理后NHEJ修復效率的變化。采用Western blot、Slot blot或免疫熒光方法檢測DNA損傷修復通路關鍵分子ATM、p-ATM、γ-H2AX、Ku80、DKA-PKcs、XLF、DNA-ligase IV和BRCA1的表達,以觀察DEB對DNA雙鏈斷裂修復通路分子表達的影響。4.NHEJ通路基因在DEB所致染色體損傷中的作用研究采用化學抑制劑Nu7026處理細胞以抑制DNA-PKcs的表達,采用Sh RNA轉染細胞以抑制Ku80的表達,通過CBMNT檢測細胞染色體損傷的變化,分析NHEJ通路在DEB所致DNA雙鏈斷裂損傷修復及NPB形成中的作用。研究結果1.DEB誘導TK6細胞增殖抑制、周期阻滯和細胞凋亡DEB可以抑制TK6細胞的活力,降低DNA的合成,具有劑量效應關系。進一步通過流式細胞儀分析發(fā)現(xiàn),DEB可以抑制TK6細胞的周期進展,使細胞分裂阻滯在G2/M期。同時,DEB處理可誘導TK6細胞凋亡,2.5~10μM DEB處理后48 h,細胞凋亡率均出現(xiàn)顯著升高(P0.01或0.001)。2.DEB誘導TK6細胞的染色體損傷及DNA雙鏈斷裂CBMNT檢測結果表明,DEB可誘導TK6細胞的微核率、核質橋率及核芽突率升高,造成細胞的染色體損傷,同時引起核分裂指數(shù)降低,抑制細胞增殖。此外,Western Blot檢測發(fā)現(xiàn),10μM DEB處理細胞后,DNA雙鏈斷裂標志物γ-H2AX蛋白的表達隨著時間的延長,先升高后降低,總體維持在一個高表達的水平。免疫熒光檢測也證實,10μM DEB處理可誘導細胞γ-H2AX的大量表達。上述結果提示,DEB可造成TK6細胞的DNA雙鏈斷裂及染色體損傷。3.DEB影響HR和NHEJ修復效率及相關基因的表達采用含有p DR-GFP底物的細胞檢測DEB對HR修復效率的影響,結果顯示,DEB處理24 h后反映HR修復的熒光陽性細胞比例顯著降低,并存在劑量效應關系,提示DEB可降低細胞的HR修復效率。體外NHEJ修復檢測結果表明,隨著DEB處理濃度增加,線性化質粒DNA連接產(chǎn)物明顯增加,提示DEB可提高DNA雙鏈斷裂的NHEJ修復效率。此外,對DNA雙鏈斷裂損傷修復通路的蛋白表達檢測結果表明,HR通路BRCA1蛋白在DEB處理24 h時出現(xiàn)明顯降低。同時,NHEJ通路蛋白DNA-PKcs、XLF和DNA-ligase IV的表達在DEB作用后4 h或24 h后開始出現(xiàn)顯著升高。Western blot檢測未發(fā)現(xiàn)NHEJ修復通路核心蛋白之一Ku80的表達改變,但是免疫熒光和Slot blot檢測顯示DEB處理細胞后4 h,Ku80蛋白形成聚焦灶,同時與DNA結合的Ku80含量顯著升高,并存在劑量效應關系。上述結果提示DEB可降低HR修復通路效率及相關基因表達,但是提高NHEJ修復通路效率,以及相關基因表達或轉移結合到DNA的能力。4.NHEJ通路參與DEB誘導的NPB形成采用化學抑制劑和sh RNA分別抑制DNA-PKcs和Ku80蛋白,通過CBMNT,觀察NHEJ通路在DEB誘導染色體損傷和NPB形成中的作用。檢測發(fā)現(xiàn),與單純DEB處理組相比,抑制DNA-PKcs表達可使DEB誘導的微核和核芽突比率顯著升高,而NPB比率顯著降低。同時,與轉染陰性對照序列的細胞相比,在穩(wěn)定轉染Ku80 sh RNA的細胞中,DEB也能誘導微核和核芽突比率進一步升高,而NPB比率則顯著降低。這些研究結果提示,抑制DNA-PKcs或Ku80蛋白的表達可加重DEB所誘導的微核及核芽突損傷,但減低了NPB損傷的發(fā)生。這一結果也提示NHEJ通路可能參與了DEB所誘導的NPB的形成。研究結論:DEB可誘導TK6細胞的DNA雙鏈斷裂和微核、核芽突以及核質橋等染色體損傷,引起細胞增殖抑制、周期阻滯和凋亡。同時,DEB引起DNA雙鏈斷裂損傷修復通路中HR修復通路的抑制和NHEJ修復通路的激活,NHEJ修復通路參與DEB誘導的核質橋的形成。
[Abstract]:Background 1,3-butadiene (BD) is an important industrial raw material used in synthetic rubber, plastics and other petrochemical and manufacturing industries. The United States ranks BD as one of the top 40 important chemicals, with a wide range of applications and a large number of occupational contacts. BD is not only a kind of occupational exposure pollutant, but also an important environmental air pollutant. Its hazard range has expanded from professional workers to the general population. BD can induce the formation of multiple organ tumors in rodents. Population studies also show the incidence of leukemia. The carcinogenic effects of BD are mainly due to the epoxidation products produced by metabolism in vivo: 1,2-epoxy-3-butene (EB), 1,2-dihydroxy-3,4-epoxy-butene (EBD) and 1,2:3,4-dioxy-butene (DEB). Previous studies have found that BD and its metabolites can induce DNA double-strand breaks and chromosome damage, but the repair mechanism of BD-induced DNA double-strand breaks is not clear. NPB is a marker of chromosomal damage and an important link in cell carcinogenesis. Studying the mechanism of BD-induced NPB formation will provide important basis for clarifying the carcinogenic process of BD, preventing and reducing the chromosomal damage induced by BD and the occurrence of human tumors. After DNA double-strand breakage, it was repaired by non-homologous end-joining (NHEJ) and other erroneous repairs to form dicentric chromosomes, which were towed to two daughter nuclei at the late stage of cell division. In order to further verify the role of the repair pathway in the formation of DEB-induced NPB, we studied the effects of DEB on DNA double-strand break repair pathway in human lymphoblasts under the conditions of clear induction of DNA double-strand break and chromosome damage. The cytotoxic effects of DEB on human lymphoblasts TK6 cells were studied. MTS and Ed U methods were used to detect the effects of DEB on cell viability and proliferation. Flow cytometry was used to analyze the cytotoxic effects of DEB on human lymphoblasts TK6 cells. The effect of DEB on cell cycle and apoptosis was detected by color assay. 2. The cytokinesis block micronucleus test (CBMNT) was used to detect the damage of DEB to cell chromosome (micronucleus rate, nucleoplasmic bridge rate, nucleus germination rate and mitotic index); Western blot and immunofluorescence were used to detect DNA double-strand break markers (gamma-cleavage index). The effect of DEB on repair of DNA double strand breakage was detected by MCF-7 cells containing the substrate of HR repair efficiency test report, and the change of HR repair efficiency after DEB treatment was detected. Western blot, Slot blot or immunofluorescence were used to detect the expression of ATM, p-ATM, gamma-H2AX, Ku80, DKA-PKcs, XLF, DNA-ligase IV and BRCA1 in DNA damage repair pathway. 4. The role of NHEJ pathway gene in chromosome damage induced by DEB was studied by chemical inhibition. Nu7026 treated cells to inhibit the expression of DNA-PKcs, Sh RNA transfected cells to inhibit the expression of Ku80, and CBMNT was used to detect the changes of chromosome damage. The role of NHEJ pathway in DNA double-strand breakage repair and NPB formation induced by DEB was analyzed. Results 1. DEB induced TK6 cells proliferation inhibition, cell cycle arrest and cell apoptosis D EB could inhibit the activity of TK6 cells and decrease the synthesis of DNA in a dose-dependent manner. Further analysis by flow cytometry showed that DEB could inhibit the cycle progression of TK6 cells and block cell division in G2/M phase. At the same time, DEB treatment could induce apoptosis of TK6 cells, and the apoptosis rate increased significantly 48 hours after 2.5-10 Mu DEB treatment (P 0.05). DEB-induced chromosomal damage and DNA double strand breakage CBMNT assay showed that DEB could induce the micronucleus rate, nucleoplasmic bridge rate and nucleus budding rate of TK6 cells to increase, resulting in chromosomal damage, while causing a decrease in mitotic index and inhibiting cell proliferation. The expression of DNA double-strand break marker gamma-H2AX protein increased first and then decreased with the prolongation of time, and remained at a high level. Immunofluorescence assay also confirmed that the treatment of 10 mu DEB could induce a large expression of gamma-H2AX in TK6 cells. The repair efficiency of HR and NHEJ and the expression of related genes were detected by cells containing P DR-GFP substrates. The results showed that the percentage of fluorescent positive cells which could reflect HR repair decreased significantly after 24 hours of treatment with DEB, and there was a dose-effect relationship, suggesting that DEB could reduce the repair efficiency of HR cells. The results showed that the linearized plasmid DNA junction products increased significantly with the increase of DEB concentration, suggesting that DEB could improve the repair efficiency of DNA double strand breaks. In addition, the expression of BRCA1 protein in DNA double strand breaks repair pathway was significantly decreased after 24 hours of DEB treatment. Western blot showed no change in the expression of Ku80, one of the core proteins of the NHEJ repair pathway. However, immunofluorescence and Slot blot showed that Ku80 protein formed a foci 4 hours after DEB treatment and the Ku80 content bound to DNA increased significantly. These results suggest that DEB can decrease the efficiency of HR repair pathway and related gene expression, but increase the efficiency of NHEJ repair pathway and the ability of related gene expression or transfer to DNA. 4. NHEJ pathway participates in the formation of DEB-induced NPB by inhibiting DNA-PKcs and Ku80 proteins by chemical inhibitors and sh RNA, respectively. CBMNT, observed the role of NHEJ pathway in DEB-induced chromosomal damage and NPB formation. Inhibition of DNA-PKcs expression significantly increased the DEB-induced micronucleus and nucleus-bud ratio, while significantly decreased the NPB ratio. Meanwhile, compared with the cells transfected with negative control sequence, the cells transfected with Ku80 sh RNA were stably transfected. These results suggest that inhibiting the expression of DNA-PKcs or Ku80 proteins may aggravate DEB-induced micronucleus and nuclear bud damage, but reduce the occurrence of NPB damage. These results also suggest that NHEJ pathway may be involved in DEB-induced NPB damage. CONCLUSION: DEB can induce DNA double strand breakage and micronucleus, nucleus bud and nucleoplasmic bridge damage in TK6 cells, which can inhibit cell proliferation, cycle arrest and apoptosis. At the same time, DEB can inhibit HR repair pathway and activate NHEJ repair pathway in DNA double strand breakage repair pathway. NHEJ repair pathway participates in DEB-induced DNA double strand breakage repair pathway. The formation of nuclear bridges.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R114
本文編號：2205902
[Abstract]:Background 1,3-butadiene (BD) is an important industrial raw material used in synthetic rubber, plastics and other petrochemical and manufacturing industries. The United States ranks BD as one of the top 40 important chemicals, with a wide range of applications and a large number of occupational contacts. BD is not only a kind of occupational exposure pollutant, but also an important environmental air pollutant. Its hazard range has expanded from professional workers to the general population. BD can induce the formation of multiple organ tumors in rodents. Population studies also show the incidence of leukemia. The carcinogenic effects of BD are mainly due to the epoxidation products produced by metabolism in vivo: 1,2-epoxy-3-butene (EB), 1,2-dihydroxy-3,4-epoxy-butene (EBD) and 1,2:3,4-dioxy-butene (DEB). Previous studies have found that BD and its metabolites can induce DNA double-strand breaks and chromosome damage, but the repair mechanism of BD-induced DNA double-strand breaks is not clear. NPB is a marker of chromosomal damage and an important link in cell carcinogenesis. Studying the mechanism of BD-induced NPB formation will provide important basis for clarifying the carcinogenic process of BD, preventing and reducing the chromosomal damage induced by BD and the occurrence of human tumors. After DNA double-strand breakage, it was repaired by non-homologous end-joining (NHEJ) and other erroneous repairs to form dicentric chromosomes, which were towed to two daughter nuclei at the late stage of cell division. In order to further verify the role of the repair pathway in the formation of DEB-induced NPB, we studied the effects of DEB on DNA double-strand break repair pathway in human lymphoblasts under the conditions of clear induction of DNA double-strand break and chromosome damage. The cytotoxic effects of DEB on human lymphoblasts TK6 cells were studied. MTS and Ed U methods were used to detect the effects of DEB on cell viability and proliferation. Flow cytometry was used to analyze the cytotoxic effects of DEB on human lymphoblasts TK6 cells. The effect of DEB on cell cycle and apoptosis was detected by color assay. 2. The cytokinesis block micronucleus test (CBMNT) was used to detect the damage of DEB to cell chromosome (micronucleus rate, nucleoplasmic bridge rate, nucleus germination rate and mitotic index); Western blot and immunofluorescence were used to detect DNA double-strand break markers (gamma-cleavage index). The effect of DEB on repair of DNA double strand breakage was detected by MCF-7 cells containing the substrate of HR repair efficiency test report, and the change of HR repair efficiency after DEB treatment was detected. Western blot, Slot blot or immunofluorescence were used to detect the expression of ATM, p-ATM, gamma-H2AX, Ku80, DKA-PKcs, XLF, DNA-ligase IV and BRCA1 in DNA damage repair pathway. 4. The role of NHEJ pathway gene in chromosome damage induced by DEB was studied by chemical inhibition. Nu7026 treated cells to inhibit the expression of DNA-PKcs, Sh RNA transfected cells to inhibit the expression of Ku80, and CBMNT was used to detect the changes of chromosome damage. The role of NHEJ pathway in DNA double-strand breakage repair and NPB formation induced by DEB was analyzed. Results 1. DEB induced TK6 cells proliferation inhibition, cell cycle arrest and cell apoptosis D EB could inhibit the activity of TK6 cells and decrease the synthesis of DNA in a dose-dependent manner. Further analysis by flow cytometry showed that DEB could inhibit the cycle progression of TK6 cells and block cell division in G2/M phase. At the same time, DEB treatment could induce apoptosis of TK6 cells, and the apoptosis rate increased significantly 48 hours after 2.5-10 Mu DEB treatment (P 0.05). DEB-induced chromosomal damage and DNA double strand breakage CBMNT assay showed that DEB could induce the micronucleus rate, nucleoplasmic bridge rate and nucleus budding rate of TK6 cells to increase, resulting in chromosomal damage, while causing a decrease in mitotic index and inhibiting cell proliferation. The expression of DNA double-strand break marker gamma-H2AX protein increased first and then decreased with the prolongation of time, and remained at a high level. Immunofluorescence assay also confirmed that the treatment of 10 mu DEB could induce a large expression of gamma-H2AX in TK6 cells. The repair efficiency of HR and NHEJ and the expression of related genes were detected by cells containing P DR-GFP substrates. The results showed that the percentage of fluorescent positive cells which could reflect HR repair decreased significantly after 24 hours of treatment with DEB, and there was a dose-effect relationship, suggesting that DEB could reduce the repair efficiency of HR cells. The results showed that the linearized plasmid DNA junction products increased significantly with the increase of DEB concentration, suggesting that DEB could improve the repair efficiency of DNA double strand breaks. In addition, the expression of BRCA1 protein in DNA double strand breaks repair pathway was significantly decreased after 24 hours of DEB treatment. Western blot showed no change in the expression of Ku80, one of the core proteins of the NHEJ repair pathway. However, immunofluorescence and Slot blot showed that Ku80 protein formed a foci 4 hours after DEB treatment and the Ku80 content bound to DNA increased significantly. These results suggest that DEB can decrease the efficiency of HR repair pathway and related gene expression, but increase the efficiency of NHEJ repair pathway and the ability of related gene expression or transfer to DNA. 4. NHEJ pathway participates in the formation of DEB-induced NPB by inhibiting DNA-PKcs and Ku80 proteins by chemical inhibitors and sh RNA, respectively. CBMNT, observed the role of NHEJ pathway in DEB-induced chromosomal damage and NPB formation. Inhibition of DNA-PKcs expression significantly increased the DEB-induced micronucleus and nucleus-bud ratio, while significantly decreased the NPB ratio. Meanwhile, compared with the cells transfected with negative control sequence, the cells transfected with Ku80 sh RNA were stably transfected. These results suggest that inhibiting the expression of DNA-PKcs or Ku80 proteins may aggravate DEB-induced micronucleus and nuclear bud damage, but reduce the occurrence of NPB damage. These results also suggest that NHEJ pathway may be involved in DEB-induced NPB damage. CONCLUSION: DEB can induce DNA double strand breakage and micronucleus, nucleus bud and nucleoplasmic bridge damage in TK6 cells, which can inhibit cell proliferation, cycle arrest and apoptosis. At the same time, DEB can inhibit HR repair pathway and activate NHEJ repair pathway in DNA double strand breakage repair pathway. NHEJ repair pathway participates in DEB-induced DNA double strand breakage repair pathway. The formation of nuclear bridges.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R114
【參考文獻】
相關期刊論文 前7條
1 田曉娟;劉海峰;柳建強;;Ku80在石英誘導細胞周期改變中作用[J];中國公共衛(wèi)生;2015年01期
2 汪海新;應明真;王梅;;TopBP1在DNA損傷修復及乳腺癌發(fā)生中的作用[J];醫(yī)學研究雜志;2012年10期
3 向夢龍;敖琳;劉晉垎;曹佳;;胞質分裂阻滯微核細胞組學試驗法的研究進展[J];癌變·畸變·突變;2012年03期
4 黎萍;許溟宇;馬青山;張捷;;靶向沉默Ku80增強順鉑誘導的人肺腺癌細胞凋亡[J];中國免疫學雜志;2012年04期
5 錢伯章;;丁二烯的技術進展與國內(nèi)外市場分析(上)[J];上�；�;2011年07期
6 劉楠;關維俊;孟會林;崔濤;李忠生;程娟;肖經(jīng)緯;王學生;李斌;;1,3-丁二烯作業(yè)工人XRCC4基因多態(tài)性與染色體損傷的關系[J];衛(wèi)生研究;2010年04期
7 虞琳;王民生;;一種多觀察終點的生物檢測方法—胞質分裂阻滯微核試驗[J];江蘇預防醫(yī)學;2007年01期
，本文編號：2205902
本文鏈接：http://sikaile.net/yixuelunwen/yufangyixuelunwen/2205902.html
最近更新
教材專著
