除草剂扑草净的细胞毒性作用及相关机制研究

发布时间：2018-05-31 05:35

本文选题：扑草净 + A549细胞　；参考：《浙江大学》2017年硕士论文

【摘要】：目的:扑草净(prometryn)属于三嗪类除草剂,与其它除草剂相比,其除草效果好,毒性低,单位使用面积用量小,在国内应用范围广,使用量大。已有研究证实体外扑草净暴露会诱导小鼠的胸腺、淋巴结和脾脏细胞发生凋亡和坏死。在食物如牛奶,紫菜、鱼虾和海参等水产品中都有较高含量的扑草净残留,同时在人体尿液、乳汁及血浆中也均能检测到扑草净的存在,但目前关于其对人类细胞毒性影响及其具体机制的体外研究还未见报道。肺部是环境污染物累积的主要器官之一,本研究以扑草净为研究对象,选用人非小细胞肺癌细胞(A549)和人支气管上皮样细胞(BEAS-2B)作为受试细胞,探讨扑草净对细胞活力、细胞周期和细胞凋亡的影响,并检测DNA损伤和胞内ROS的产生情况,以期最终明确扑草净的细胞毒性及相关机制。方法:研究通过显微镜观察不同浓度(0,25,50,100,200μM)扑草净暴露24h和48h后对A549和BEAS-2B细胞的生长状况和形态影响;采用MTT法检测扑草净对细胞增殖活力的影响;细胞经PI单染或AnnexinV-PI双染后使用流式细胞仪检测扑草净对细胞周期和细胞凋亡的影响;使用蛋白免疫印迹技术检测周期和DNA损伤修复等相关蛋白的表达及磷酸化水平;使用免疫荧光和彗星实验检测DNA损伤情况;细胞经DCFH-DA染色后,通过荧光显微镜观察胞内ROS的产生情况,使用流式细胞术对胞内ROS水平进行定量检测。结果:1.扑草净在100-200μM处理浓度范围内,显微镜下观察到A549细胞变圆,贴壁能力减弱,细胞数量减少;部分BEAS-2B细胞出现明显皱缩,上清液中漂浮细胞和细胞碎片增多。2.扑草净在200μM浓度下处理24小时、50-200μM浓度范围内处理48小时,能引起A549细胞相对存活率的显著降低;在100-200μM处理浓度范围内作用48小时后,BEAS-2B细胞相对存活率明显降低。3.扑草净在100-200μM处理浓度范围内作用48小时,能诱导A549细胞发生G1期细胞阻滞,p53、p27和p21蛋白表达升高,同时E2F1、CyclinD1、CDK4、CyclinE和CDK2蛋白的表达降低;并诱导BEAS-2B细胞发生S期阻滞,p53表达升高,CyclinA、CDK2 表达下调。4.扑草净处理后,A549细胞凋亡数量较少,但出现Bcl2表达减少,Bax表达升高;BEAS-2B细胞出现明显凋亡,Caspase9、Caspase3和PARP前体表达下降,且均出现明显的剪切体。5.高至200μM浓度的扑草净处理A549细胞也未引起其产生明显的ROS;在200μM浓度时,可诱导BEAS-2B细胞产生明显的ROS。6.扑草净在50-200μM处理浓度范围内,能明显诱导A549和BEAS-2B细胞DNA损伤,并出现DNA双链断裂(DSBs),剂量越高,DNA断裂程度越严重,彗星实验TDNA%率越高(P0.05;BEAS-2B细胞2000μM浓度组加入抗氧化剂NAC预处理后,能减轻DNA损伤。7.扑草净处理后,A549和BEAS-2B细胞内H2AX均发生明显磷酸化,A549细胞γ-H2AX荧光焦点有排出主核趋势;BEAS-2B细胞核内γ-H2AX染色呈散点或团块状均匀分布。结论:1.在50-200μM处理浓度范围内,扑草净能诱导A549细胞发生DNA损伤,并引发DNA损伤等应激反应,p53、p21及p27等相关蛋白参与周期调控,细胞周期进程被阻滞在G1期,γ-H2AX参与形成微核并修复DNA损伤,而对凋亡和胞内ROS水平无明显影响。2.在100-200μM的浓度范围内,48小时的扑草净处理对BEAS-2B具有明显的损伤作用,细胞存活率下降,细胞增殖和细胞周期进程受阻,胞内ROS生成增多,诱导DNA的氧化损伤,造成DNA双链断裂,细胞凋亡率升高。相关机制研究表明p53、Bcl2和Caspase3等相关蛋白参与了扑草净诱导的细胞凋亡调控。3.环境暴露剂量下,扑草净对人细胞毒性较低,但在分子水平上仍能观察到DNA损伤和相关蛋白的改变。应减少扑草净的接触,避免造成机体的健康危害。
[Abstract]:Objective: prometryn is a three pyrazine herbicide. Compared with other herbicides, it has good weed control effect, low toxicity, small use area, wide application and large amount of use in our country. It has been found that paracetamol exposure can induce apoptosis and necrosis of mice's thymus, lymph nodes and spleen cells. In food such as cattle A high content of paracetamol residues in aquatic products, such as milk, laver, fish and shrimp, and sea cucumbers, can also be detected in human urine, milk and plasma, but in vitro studies on its effects on human cytotoxicity and specific mechanisms are not reported. Lung is the main organ of the accumulation of environmental pollutants. 1. In this study, we used paracetamol as the research object, selected human non-small cell lung cancer cells (A549) and human bronchial epithelioid cells (BEAS-2B) as the tested cells, and explored the effect of acetamiol on cell vitality, cell cycle and cell apoptosis, and detected the damage of DNA and the production of intracellular ROS, in order to clarify the cytotoxicity of acetamiyn and the cytotoxicity of acetamiyn. Related mechanisms. Methods: the effects of 0,25,50100200 and 48h on the growth and morphology of A549 and BEAS-2B cells were investigated by microscopic observation of 24h and 48h with different concentrations (M). The effect of acetamol on cell proliferation was detected by MTT, and the cells were detected by flow cytometer after PI single staining or AnnexinV-PI double staining. The effect of cell cycle and apoptosis; using protein immunoblotting technique to detect the expression and phosphorylation level of DNA damage repair and other related proteins; use immunofluorescence and comet assay to detect DNA damage; cells were stained by DCFH-DA to observe the production of intracellular ROS by fluorescence microscopy, using flow cytometry Quantitative detection of intracellular ROS level. Results: 1. paracetamol was within the concentration range of 100-200 mu M. Under the microscope, the A549 cells became round, the adhesion ability was reduced, the number of cells decreased, the number of BEAS-2B cells decreased obviously, and the floating cells and cell fragments in the supernatant were increased by.2. acetamiyrum for 24 hours and 50-200 Mu under the concentration of 200 mu M. After 48 hours of M concentration, the relative survival rate of A549 cells decreased significantly. After 48 hours in the 100-200 M concentration range, the relative survival rate of BEAS-2B cells decreased significantly for 48 hours in the concentration range of 100-200 mu M, which could induce G1 phase cell block in A549 cells, p53, p27 and p21 protein tables. At the same time, the expression of E2F1, CyclinD1, CDK4, CyclinE and CDK2 decreased, and the BEAS-2B cells were induced by S block, the expression of p53 increased, CyclinA, CDK2 expression decreased, but the number of apoptotic A549 cells was less. The expression of P precursor decreased, and the A549 cells with obvious shear body.5. high to 200 M did not cause obvious ROS. At the concentration of 200 mu M, the BEAS-2B cells could induce the obvious ROS.6. acetoacetum in the concentration range of 50-200 u M, which could obviously induce DNA damage of A549 and BEAS-2B cells. The higher the dose of DSBs, the higher the dose, the more serious the DNA fracture, the higher the TDNA% rate of the comet experiment (P0.05; after the BEAS-2B cell 2000 mu M concentration group added to the antioxidant NAC pretreatment, the DNA damage.7. paracetamol treatment, A549 and BEAS-2B cells were obviously phosphorylated. The staining of gamma -H2AX in the nucleus of B was distributed uniformly. Conclusion: 1. in the concentration range of 50-200 micron M, acetamol can induce DNA damage in A549 cells and induce DNA damage and other stress reactions. P53, p21 and p27 related proteins participate in the periodic regulation, and the cell cycle progression is blocked in G1, and gamma -H2AX participates in the formation of micronucleus and repair D. NA damage, but no obvious effect on apoptosis and intracellular ROS level of.2. in the concentration range of 100-200 mu M, 48 hours of paracetamol treatment had obvious damage to BEAS-2B, cell survival rate decreased, cell proliferation and cell cycle process blocked, intracellular ROS production increased, induced DNA oxidative damage, resulting in DNA double strand breakage, cell apoptosis rate The related mechanism study showed that p53, Bcl2, Caspase3 and other related proteins participated in the.3. environment exposure dose induced by paracetamol, and the toxicity of paracetamol to human cells was low, but at the molecular level, the DNA damage and the changes of related proteins were still observed. Harm.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114

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