本文選題：撲草凈 + A549細胞�。� 參考：《浙江大學》2017年碩士論文

【摘要】：目的:撲草凈(prometryn)屬于三嗪類除草劑,與其它除草劑相比,其除草效果好,毒性低,單位使用面積用量小,在國內應用范圍廣,使用量大。已有研究證實體外撲草凈暴露會誘導小鼠的胸腺、淋巴結和脾臟細胞發(fā)生凋亡和壞死。在食物如牛奶,紫菜、魚蝦和海參等水產品中都有較高含量的撲草凈殘留,同時在人體尿液、乳汁及血漿中也均能檢測到撲草凈的存在,但目前關于其對人類細胞毒性影響及其具體機制的體外研究還未見報道。肺部是環(huán)境污染物累積的主要器官之一,本研究以撲草凈為研究對象,選用人非小細胞肺癌細胞(A549)和人支氣管上皮樣細胞(BEAS-2B)作為受試細胞,探討撲草凈對細胞活力、細胞周期和細胞凋亡的影響,并檢測DNA損傷和胞內ROS的產生情況,以期最終明確撲草凈的細胞毒性及相關機制。方法:研究通過顯微鏡觀察不同濃度(0,25,50,100,200μM)撲草凈暴露24h和48h后對A549和BEAS-2B細胞的生長狀況和形態(tài)影響;采用MTT法檢測撲草凈對細胞增殖活力的影響;細胞經PI單染或AnnexinV-PI雙染后使用流式細胞儀檢測撲草凈對細胞周期和細胞凋亡的影響;使用蛋白免疫印跡技術檢測周期和DNA損傷修復等相關蛋白的表達及磷酸化水平;使用免疫熒光和彗星實驗檢測DNA損傷情況;細胞經DCFH-DA染色后,通過熒光顯微鏡觀察胞內ROS的產生情況,使用流式細胞術對胞內ROS水平進行定量檢測。結果:1.撲草凈在100-200μM處理濃度范圍內,顯微鏡下觀察到A549細胞變圓,貼壁能力減弱,細胞數量減少;部分BEAS-2B細胞出現(xiàn)明顯皺縮,上清液中漂浮細胞和細胞碎片增多。2.撲草凈在200μM濃度下處理24小時、50-200μM濃度范圍內處理48小時,能引起A549細胞相對存活率的顯著降低;在100-200μM處理濃度范圍內作用48小時后,BEAS-2B細胞相對存活率明顯降低。3.撲草凈在100-200μM處理濃度范圍內作用48小時,能誘導A549細胞發(fā)生G1期細胞阻滯,p53、p27和p21蛋白表達升高,同時E2F1、CyclinD1、CDK4、CyclinE和CDK2蛋白的表達降低;并誘導BEAS-2B細胞發(fā)生S期阻滯,p53表達升高,CyclinA、CDK2 表達下調。4.撲草凈處理后,A549細胞凋亡數量較少,但出現(xiàn)Bcl2表達減少,Bax表達升高;BEAS-2B細胞出現(xiàn)明顯凋亡,Caspase9、Caspase3和PARP前體表達下降,且均出現(xiàn)明顯的剪切體。5.高至200μM濃度的撲草凈處理A549細胞也未引起其產生明顯的ROS;在200μM濃度時,可誘導BEAS-2B細胞產生明顯的ROS。6.撲草凈在50-200μM處理濃度范圍內,能明顯誘導A549和BEAS-2B細胞DNA損傷,并出現(xiàn)DNA雙鏈斷裂(DSBs),劑量越高,DNA斷裂程度越嚴重,彗星實驗TDNA%率越高(P0.05;BEAS-2B細胞2000μM濃度組加入抗氧化劑NAC預處理后,能減輕DNA損傷。7.撲草凈處理后,A549和BEAS-2B細胞內H2AX均發(fā)生明顯磷酸化,A549細胞γ-H2AX熒光焦點有排出主核趨勢;BEAS-2B細胞核內γ-H2AX染色呈散點或團塊狀均勻分布。結論:1.在50-200μM處理濃度范圍內,撲草凈能誘導A549細胞發(fā)生DNA損傷,并引發(fā)DNA損傷等應激反應,p53、p21及p27等相關蛋白參與周期調控,細胞周期進程被阻滯在G1期,γ-H2AX參與形成微核并修復DNA損傷,而對凋亡和胞內ROS水平無明顯影響。2.在100-200μM的濃度范圍內,48小時的撲草凈處理對BEAS-2B具有明顯的損傷作用,細胞存活率下降,細胞增殖和細胞周期進程受阻,胞內ROS生成增多,誘導DNA的氧化損傷,造成DNA雙鏈斷裂,細胞凋亡率升高。相關機制研究表明p53、Bcl2和Caspase3等相關蛋白參與了撲草凈誘導的細胞凋亡調控。3.環(huán)境暴露劑量下,撲草凈對人細胞毒性較低,但在分子水平上仍能觀察到DNA損傷和相關蛋白的改變。應減少撲草凈的接觸,避免造成機體的健康危害。
[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.
【學位授予單位】：浙江大學
【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4條

1 高云,何芳;接觸撲草凈工人眼部影響的調查[J];眼外傷職業(yè)眼病雜志.附眼科手術;2000年02期

2 李淑娟;陳冬東;李曉娟;安娟;蔡會霞;李建中;唐英章;;氣相色譜-質譜法測定食品中撲草凈的殘留量[J];中國衛(wèi)生檢驗雜志;2007年12期

3 高利娜;劉國杰;劉俊亭;祝娟;王春媛;;固相微萃取結合氣相色譜法測定水樣中痕量撲草凈[J];中國法醫(yī)學雜志;2013年06期

4 ;[J];;年期

相關會議論文 前4條

1 覃宇成;陳詩佩;李玉萍;;復合型除草殺蟲劑雙滅靈中甲拌磷、撲草凈、丁草胺的氣相色譜分析[A];中國化工學會農藥專業(yè)委員會第八屆年會論文集[C];1996年

2 曹春田;臧學斌;禹成松;梅紅;劉新峰;;40%乙草胺·撲草凈懸浮乳劑防除棉花田雜草田間藥效試驗研究[A];河南省植保學會第九次、河南省昆蟲學會第八次、河南省植病學會第三次會員代表大會暨學術討論會論文集[C];2009年

3 張香云;張彥民;員白燕;張凱遠;;72%甲硫嘧黃隆·撲草凈WDG防除小麥田雜草藥效試驗[A];河南省植物保護研究進展Ⅱ（上）[C];2007年

4 周際海;孫向武;李輝信;;食細菌線蟲的馴化及生長特性初步研究[A];面向未來的土壤科學（中冊）——中國土壤學會第十二次全國會員代表大會暨第九屆海峽兩岸土壤肥料學術交流研討會論文集[C];2012年

相關重要報紙文章 前2條

1 龐林 蔡炳江;排除一項風險 救活一家企業(yè)[N];中國國門時報;2012年

2 本期專家 中化化肥高級顧問、中國農業(yè)大學教授 王興仁　肖悅巖;肥料與農藥混合注意事項[N];農民日報;2009年

相關博士學位論文 前1條

1 姜蕾;有機質對除草劑撲草凈環(huán)境行為的影響研究[D];南京農業(yè)大學;2011年

相關碩士學位論文 前9條

1 王亞萍;農藥、塑化劑與蛋白質的結合機理研究[D];南昌大學;2015年

2 梁棟;撲草凈降解菌DY-1的篩選、鑒定及特性的研究[D];東北農業(yè)大學;2016年

3 劉軍委;一株撲草凈降解菌的分離鑒定、降解特性及其代謝途徑研究[D];安徽農業(yè)大學;2016年

4 劉巧云;除草劑撲草凈的細胞毒性作用及相關機制研究[D];浙江大學;2017年

5 伍長春;甲硫嘧磺隆混配技術與甲硫嘧磺隆·撲草凈水分散粒劑研究[D];湖南農業(yè)大學;2006年

6 曹軍;除草劑撲草凈在土壤環(huán)境中的行為研究[D];南京農業(yè)大學;2008年

7 尹忠達;撲草凈與VA草酮對野慈姑的混用效應及水稻田應用技術研究[D];東北農業(yè)大學;2014年

8 周秉彥;電化學產生H_2O_2助Fe/TiO_2可見光催化降解水中撲草凈的研究[D];鄭州大學;2014年

9 江海瀾;除草劑與植物生長調節(jié)劑互作對棉田龍葵的影響及生理機制研究[D];石河子大學;2013年

，

本文編號：1958487