1、甲醛誘導(dǎo)MRC-5細(xì)胞適應(yīng)性反應(yīng)蛋白質(zhì)研究                 作者：李習(xí)藝，莊志雄，劉建軍，楊曉華，衛(wèi)秦芝，黃海燕【關(guān)鍵詞】  甲醛；適應(yīng)性反應(yīng)；蛋白質(zhì)組摘要： 目的   研究甲醛誘導(dǎo)真核細(xì)胞適應(yīng)性反應(yīng)及其可能的機(jī)制。方法   建立甲醛刺激人胚肺成纖維細(xì)胞(MRC-5)適應(yīng)性反應(yīng)模型；采用AlamarBlue還原率檢測細(xì)胞增殖。雙向凝膠電泳分離細(xì)胞總蛋白，圖像分析后選取差異蛋白點進(jìn)行

2、質(zhì)譜鑒定。結(jié)果   AlamarBlue檢測結(jié)果表明，低劑量甲醛預(yù)刺激可誘導(dǎo)細(xì)胞適應(yīng)性反應(yīng)。雙向凝膠電泳結(jié)果顯示，甲醛刺激后19個蛋白斑點發(fā)生變化，肽指紋圖譜及肽序列標(biāo)簽鑒定了其中13個蛋白斑點，已鑒定的蛋白包括二磷酸核苷酸激酶、延長因子1-，磷酸丙糖異構(gòu)酶、60S酸性核糖體蛋白P0、75kDa熱休克蛋白、70kD熱休克蛋白樣結(jié)合蛋白、鈣依賴磷脂結(jié)合蛋白I、假想蛋白FLJ34423、微管-肌動蛋白交叉連接因子1、核纖層蛋白B2、ATP合成酶鏈、蛋白酶體亞基6，這些蛋白功能涉及轉(zhuǎn)錄調(diào)節(jié)、蛋白折疊、信號傳導(dǎo)、能量代謝、細(xì)胞骨架等各個方面。結(jié)論   低劑量甲醛

3、可誘導(dǎo)MRC-5細(xì)胞適應(yīng)性反應(yīng)，多種蛋白參與細(xì)胞的適應(yīng)性反應(yīng)調(diào)控。關(guān)鍵詞： 甲醛；適應(yīng)性反應(yīng)；蛋白質(zhì)組Proteomie analysis of adaptive response induced by low concentration of formaldehyde in human embryonic fibroblasts cells   Abstract： Objective   To study the adaptive response induced by formaldehyde(FA) on eukaryocyte and its p

4、ossible mechanism.Methods   After FA treatment，AlamarBlue reducing rate was observed to determine the cell proliferation.The total cellular proteins were separated using two-dimensional gel electrophoresis and visualized by sliver staining.Gels from different batches of control and FA-trea

5、ted MRC-5 cells were analyzed for quantitative spot comparisons with the ImageMaster 2D platinum 5.0 software.The differentially expressed prorein spots were picked and digested in gel then identified by tandem mass spectrum.Results   The results of AlamarBlue reducing rate showed that low

6、 concentration of FA induced adaptive response on MRC-5 cells.Nineteen spots were changed significantly after FA treatment.Thirteen proteins were identified by peptide mass fingerprinting or peptide sequence analysis，including putative nucleoside diphosphate kinase，elongation factor 1-gamma，trioseph

7、osphate isomerase，60S acidic ribosomal protein P0，heat shock protein 75 kDa,similar to heat shock 70kD protein binding protein，annexin I，hypothetical protein FLJ34423，microtubule-actin crosslinking factor 1，lamin B2，ATP synthase alpha chain，mitochondrial precursor，proteasome subunit alpha type 6.The

8、se identified proteins involved in energy metabolism，translation and RNA processing,protein folding，redox regulation,cell structure and cell signaling.Conclusion   Adaptive response can be induced by low concentration of FA on MRC-5 cells and cellular adaptation is a complex process involv

9、ing in a modulation of diverse cellular functions.Key words： formaldehyde；adaptive response；proteomics      適應(yīng)性反應(yīng)（adaptive response）是機(jī)體受到低劑量環(huán)境因子預(yù)刺激后對后續(xù)高劑量的刺激產(chǎn)生耐受的現(xiàn)象。研究表明1，許多環(huán)境刺激因子低劑量接觸可誘導(dǎo)適應(yīng)性反應(yīng)。但適應(yīng)性反應(yīng)是否為自然界普遍存在的現(xiàn)象，適應(yīng)性反應(yīng)的機(jī)制還未闡明。甲醛(FA)是常見的環(huán)境化學(xué)污染物，早期研究發(fā)現(xiàn)，機(jī)體接觸低劑量甲醛可能誘導(dǎo)產(chǎn)生某種耐受與適應(yīng)現(xiàn)象

10、，但尚未對其機(jī)制進(jìn)行探討2，3。本實驗利用建立的甲醛適應(yīng)性反應(yīng)細(xì)胞模型，采用蛋白質(zhì)組學(xué)技術(shù)研究了甲醛刺激后人胚肺成纖維細(xì)胞蛋白質(zhì)表達(dá)差異，并用串聯(lián)質(zhì)譜鑒定差異蛋白，為了解甲醛誘導(dǎo)細(xì)胞適應(yīng)性反應(yīng)的分子機(jī)制提供依據(jù)。1   材料與方法11   材料   甲醛(上海標(biāo)準(zhǔn)試劑）、硫脲(美國Sigm公司)；等電聚焦膠條(13cm pH47)、尿素、二巰基蘇糖醇(瑞典Amersham公司)；丙基磺酸鹽、碘乙酰氨(美國USP公司)；胰蛋白酶(測序級)、丙烯酰胺(美國Promega公司)；AlamarBlue染料(美國Biosource公司)；其余試

11、劑采用國產(chǎn)分析純。12   方法121   細(xì)胞培養(yǎng)   MRC-5細(xì)胞來源于成都腫瘤研究所，培養(yǎng)于改良的伊格培養(yǎng)基(DMEM)，含10胎牛血清，100kU/L青霉素，100mg/L鏈霉素。FA臨用前用無血清DMEM配制。122   AlamarBlue檢測細(xì)胞增殖   將細(xì)胞接種于96孔板，至對數(shù)生長期，按實驗方案分別加入適應(yīng)性反應(yīng)劑量(10mol/L)及攻擊劑量(500mol/L)的甲醛，對照組分別加入相同體積的無血清DMEM培養(yǎng)基，培養(yǎng)結(jié)束前4h每孔加入10l染料，繼續(xù)培養(yǎng)4h用酶標(biāo)儀測吸

12、光度值計算AlamarBlue還原率。123   雙向凝膠電泳   參照Amersham公司技術(shù)手冊及本實驗室改進(jìn)的技術(shù)方法4，上樣量90g，上樣體積250l，等電聚焦4200048000vhs，聚焦時間1921h。樣品收集方案：FA染毒低劑量組為10mol/L刺激12h，高劑量組為500mol/L FA刺激5h，適應(yīng)組為10mol/L FA預(yù)刺激12h后500mol/L FA刺激5h，空白對照組加入相應(yīng)體積無血清DMEM，收集不同批次的細(xì)胞重復(fù)至少3次。124   圖像采集和分析   染色后的雙向電泳凝膠用的圖像掃描儀掃描