1,2:3,4-二环氧丁烷诱导细胞DNA双链断裂损伤及其修复机制研究
发布时间:2018-08-26 19:16
【摘要】:研究背景1,3-丁二烯(1,3-butadiene,BD)是一种重要的工业原料,用于合成橡胶、塑料等石油化工和制造行业。美国将BD列为前40位重要的化学品之一,应用范围广,职业接触人群多。此外,汽车尾气、香烟烟雾及食用油油烟中也发现BD普遍存在,属于室内具有慢性空气危害的9种优先污染物之一。因此BD不仅是一种职业暴露污染物,更是一种重要的环境空气污染物,其危害范围已从职业工人扩展到普通人群。BD可以诱导啮齿动物多器官肿瘤的形成,人群研究也显示出白血病发病相关的阳性结果,因而被IARC和EPA列为确定的人类致癌物(1组)。BD的致癌效应主要来自于体内代谢产生的环氧化产物:1,2-环氧-3-丁烯(EB),1,2-二羟基-3,4-环氧丁烯(EBD)和1,2:3,4-二环氧丁烯(DEB)。上述环氧化产物均能与DNA形成加合物,造成遗传物质的损伤,其中DEB的活性最强,因而目前对BD致癌机制研究多聚焦于阐明其遗传损伤效应。既往研究发现BD及其代谢物可以诱导DNA双链断裂和染色体损伤,但是BD诱导DNA双链断裂损伤后的修复机制并不清楚。本课题组前期在人群研究中发现BD暴露与核质桥(NPB)比例升高相关。NPB是染色体损伤的标志和细胞癌变的重要环节,研究BD诱导NPB形成的机制,将为阐明BD的致癌作用过程,预防和减少BD所诱导的染色体损伤和人群肿瘤的发生提供重要依据。有研究提示,NPB的形成可能与DNA双链断裂后,经非同源末端连接(NHEJ)等错误修复形成双着丝粒染色体,在细胞分裂后期分别牵引到两个子细胞核有关。由此我们推测,BD及其代谢物诱导DNA双链断裂后,可能通过影响主要的DNA双链断裂修复通路,即HR和NHEJ修复通路,造成错误修复效率的提高,从而引起NPB形成。因此,本课题以BD活性代谢物DEB为对象,在明确其诱导人淋巴母细胞DNA双链断裂和染色体损伤的条件下,研究DEB对DNA双链断裂修复通路的影响,进一步验证修复通路在DEB致NPB形成中的作用,为深入探讨BD的遗传损伤机制提供依据。研究内容1.DEB的细胞毒性效应研究建立DEB染毒处理的人淋巴母细胞TK6细胞模型模型。分别采用MTS和Ed U方法检测DEB对细胞活性和增殖能力的影响。采用流式细胞仪分析技术,分别以PI和Annix V-FITC/PI染色检测DEB对细胞周期和细胞凋亡的影响。2.DEB诱导细胞遗传损伤的检测采用胞质分裂阻滞微核试验(CBMNT)分析DEB对细胞染色体的损伤(微核率、核质桥率、核芽突率及核分裂指数);采用Western blot和免疫荧光技术,检测DNA双链断裂标志物(γ-H2AX)的表达情况。3.DEB对细胞DNA双链断裂损伤修复的影响采用含有HR修复效率检测报告底物的MCF-7细胞,检测DEB处理后HR修复效率的变化。采用Cell-free方法,提取细胞总蛋白,与32P标记的线性质粒共同作用,检测DEB处理后NHEJ修复效率的变化。采用Western blot、Slot blot或免疫荧光方法检测DNA损伤修复通路关键分子ATM、p-ATM、γ-H2AX、Ku80、DKA-PKcs、XLF、DNA-ligase IV和BRCA1的表达,以观察DEB对DNA双链断裂修复通路分子表达的影响。4.NHEJ通路基因在DEB所致染色体损伤中的作用研究采用化学抑制剂Nu7026处理细胞以抑制DNA-PKcs的表达,采用Sh RNA转染细胞以抑制Ku80的表达,通过CBMNT检测细胞染色体损伤的变化,分析NHEJ通路在DEB所致DNA双链断裂损伤修复及NPB形成中的作用。研究结果1.DEB诱导TK6细胞增殖抑制、周期阻滞和细胞凋亡DEB可以抑制TK6细胞的活力,降低DNA的合成,具有剂量效应关系。进一步通过流式细胞仪分析发现,DEB可以抑制TK6细胞的周期进展,使细胞分裂阻滞在G2/M期。同时,DEB处理可诱导TK6细胞凋亡,2.5~10μM DEB处理后48 h,细胞凋亡率均出现显著升高(P0.01或0.001)。2.DEB诱导TK6细胞的染色体损伤及DNA双链断裂CBMNT检测结果表明,DEB可诱导TK6细胞的微核率、核质桥率及核芽突率升高,造成细胞的染色体损伤,同时引起核分裂指数降低,抑制细胞增殖。此外,Western Blot检测发现,10μM DEB处理细胞后,DNA双链断裂标志物γ-H2AX蛋白的表达随着时间的延长,先升高后降低,总体维持在一个高表达的水平。免疫荧光检测也证实,10μM DEB处理可诱导细胞γ-H2AX的大量表达。上述结果提示,DEB可造成TK6细胞的DNA双链断裂及染色体损伤。3.DEB影响HR和NHEJ修复效率及相关基因的表达采用含有p DR-GFP底物的细胞检测DEB对HR修复效率的影响,结果显示,DEB处理24 h后反映HR修复的荧光阳性细胞比例显著降低,并存在剂量效应关系,提示DEB可降低细胞的HR修复效率。体外NHEJ修复检测结果表明,随着DEB处理浓度增加,线性化质粒DNA连接产物明显增加,提示DEB可提高DNA双链断裂的NHEJ修复效率。此外,对DNA双链断裂损伤修复通路的蛋白表达检测结果表明,HR通路BRCA1蛋白在DEB处理24 h时出现明显降低。同时,NHEJ通路蛋白DNA-PKcs、XLF和DNA-ligase IV的表达在DEB作用后4 h或24 h后开始出现显著升高。Western blot检测未发现NHEJ修复通路核心蛋白之一Ku80的表达改变,但是免疫荧光和Slot blot检测显示DEB处理细胞后4 h,Ku80蛋白形成聚焦灶,同时与DNA结合的Ku80含量显著升高,并存在剂量效应关系。上述结果提示DEB可降低HR修复通路效率及相关基因表达,但是提高NHEJ修复通路效率,以及相关基因表达或转移结合到DNA的能力。4.NHEJ通路参与DEB诱导的NPB形成采用化学抑制剂和sh RNA分别抑制DNA-PKcs和Ku80蛋白,通过CBMNT,观察NHEJ通路在DEB诱导染色体损伤和NPB形成中的作用。检测发现,与单纯DEB处理组相比,抑制DNA-PKcs表达可使DEB诱导的微核和核芽突比率显著升高,而NPB比率显著降低。同时,与转染阴性对照序列的细胞相比,在稳定转染Ku80 sh RNA的细胞中,DEB也能诱导微核和核芽突比率进一步升高,而NPB比率则显著降低。这些研究结果提示,抑制DNA-PKcs或Ku80蛋白的表达可加重DEB所诱导的微核及核芽突损伤,但减低了NPB损伤的发生。这一结果也提示NHEJ通路可能参与了DEB所诱导的NPB的形成。研究结论:DEB可诱导TK6细胞的DNA双链断裂和微核、核芽突以及核质桥等染色体损伤,引起细胞增殖抑制、周期阻滞和凋亡。同时,DEB引起DNA双链断裂损伤修复通路中HR修复通路的抑制和NHEJ修复通路的激活,NHEJ修复通路参与DEB诱导的核质桥的形成。
[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.
【学位授予单位】:第三军医大学
【学位级别】:硕士
【学位授予年份】:2017
【分类号】:R114
本文编号: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.
【学位授予单位】:第三军医大学
【学位级别】:硕士
【学位授予年份】:2017
【分类号】:R114
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