本文選題：撲草凈 + A549細(xì)胞��； 參考：《浙江大學(xué)》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(nèi)應(yīng)用范圍廣,使用量大。已有研究證實(shí)體外撲草凈暴露會(huì)誘導(dǎo)小鼠的胸腺、淋巴結(jié)和脾臟細(xì)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(chǎn)品中都有較高含量的撲草凈殘留,同時(shí)在人體尿液、乳汁及血漿中也均能檢測(cè)到撲草凈的存在,但目前關(guān)于其對(duì)人類細(xì)胞毒性影響及其具體機(jī)制的體外研究還未見報(bào)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(duì)象,選用人非小細(xì)胞肺癌細(xì)胞(A549)和人支氣管上皮樣細(xì)胞(BEAS-2B)作為受試細(xì)胞,探討撲草凈對(duì)細(xì)胞活力、細(xì)胞周期和細(xì)胞凋亡的影響,并檢測(cè)DNA損傷和胞內(nèi)ROS的產(chǎn)生情況,以期最終明確撲草凈的細(xì)胞毒性及相關(guān)機(jī)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(duì)A549和BEAS-2B細(xì)胞的生長狀況和形態(tài)影響;采用MTT法檢測(cè)撲草凈對(duì)細(xì)胞增殖活力的影響;細(xì)胞經(jīng)PI單染或AnnexinV-PI雙染后使用流式細(xì)胞儀檢測(cè)撲草凈對(duì)細(xì)胞周期和細(xì)胞凋亡的影響;使用蛋白免疫印跡技術(shù)檢測(cè)周期和DNA損傷修復(fù)等相關(guān)蛋白的表達(dá)及磷酸化水平;使用免疫熒光和彗星實(shí)驗(yàn)檢測(cè)DNA損傷情況;細(xì)胞經(jīng)DCFH-DA染色后,通過熒光顯微鏡觀察胞內(nèi)ROS的產(chǎn)生情況,使用流式細(xì)胞術(shù)對(duì)胞內(nèi)ROS水平進(jìn)行定量檢測(cè)。結(jié)果:1.撲草凈在100-200μM處理濃度范圍內(nèi),顯微鏡下觀察到A549細(xì)胞變圓,貼壁能力減弱,細(xì)胞數(shù)量減少;部分BEAS-2B細(xì)胞出現(xiàn)明顯皺縮,上清液中漂浮細(xì)胞和細(xì)胞碎片增多。2.撲草凈在200μM濃度下處理24小時(shí)、50-200μM濃度范圍內(nèi)處理48小時(shí),能引起A549細(xì)胞相對(duì)存活率的顯著降低;在100-200μM處理濃度范圍內(nèi)作用48小時(shí)后,BEAS-2B細(xì)胞相對(duì)存活率明顯降低。3.撲草凈在100-200μM處理濃度范圍內(nèi)作用48小時(shí),能誘導(dǎo)A549細(xì)胞發(fā)生G1期細(xì)胞阻滯,p53、p27和p21蛋白表達(dá)升高,同時(shí)E2F1、CyclinD1、CDK4、CyclinE和CDK2蛋白的表達(dá)降低;并誘導(dǎo)BEAS-2B細(xì)胞發(fā)生S期阻滯,p53表達(dá)升高,CyclinA、CDK2 表達(dá)下調(diào)。4.撲草凈處理后,A549細(xì)胞凋亡數(shù)量較少,但出現(xiàn)Bcl2表達(dá)減少,Bax表達(dá)升高;BEAS-2B細(xì)胞出現(xiàn)明顯凋亡,Caspase9、Caspase3和PARP前體表達(dá)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(xì)胞也未引起其產(chǎn)生明顯的ROS;在200μM濃度時(shí),可誘導(dǎo)BEAS-2B細(xì)胞產(chǎn)生明顯的ROS。6.撲草凈在50-200μM處理濃度范圍內(nèi),能明顯誘導(dǎo)A549和BEAS-2B細(xì)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(yán)重,彗星實(shí)驗(yàn)TDNA%率越高(P0.05;BEAS-2B細(xì)胞2000μM濃度組加入抗氧化劑NAC預(yù)處理后,能減輕DNA損傷。7.撲草凈處理后,A549和BEAS-2B細(xì)胞內(nèi)H2AX均發(fā)生明顯磷酸化,A549細(xì)胞γ-H2AX熒光焦點(diǎn)有排出主核趨勢(shì);BEAS-2B細(xì)胞核內(nèi)γ-H2AX染色呈散點(diǎn)或團(tuán)塊狀均勻分布。結(jié)論:1.在50-200μM處理濃度范圍內(nèi),撲草凈能誘導(dǎo)A549細(xì)胞發(fā)生DNA損傷,并引發(fā)DNA損傷等應(yīng)激反應(yīng),p53、p21及p27等相關(guān)蛋白參與周期調(diào)控,細(xì)胞周期進(jìn)程被阻滯在G1期,γ-H2AX參與形成微核并修復(fù)DNA損傷,而對(duì)凋亡和胞內(nèi)ROS水平無明顯影響。2.在100-200μM的濃度范圍內(nèi),48小時(shí)的撲草凈處理對(duì)BEAS-2B具有明顯的損傷作用,細(xì)胞存活率下降,細(xì)胞增殖和細(xì)胞周期進(jìn)程受阻,胞內(nèi)ROS生成增多,誘導(dǎo)DNA的氧化損傷,造成DNA雙鏈斷裂,細(xì)胞凋亡率升高。相關(guān)機(jī)制研究表明p53、Bcl2和Caspase3等相關(guān)蛋白參與了撲草凈誘導(dǎo)的細(xì)胞凋亡調(diào)控。3.環(huán)境暴露劑量下,撲草凈對(duì)人細(xì)胞毒性較低,但在分子水平上仍能觀察到DNA損傷和相關(guān)蛋白的改變。應(yīng)減少撲草凈的接觸,避免造成機(jī)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(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R114

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