腹泻性贝类毒素细胞检测方法的研究
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摘要
腹泻性贝类毒素(diarrhetic shellfish poisoning,DSP)是以大田软海绵酸(okadaic acid,OA)及其衍生物为代表、摄食后可产生以腹泻为主要特征的存在于贝类体内的海洋生物毒性物质的总称。OA能够促进肿瘤形成,在极低浓度范围就可对人类健康造成潜在的威胁,其慢性毒性效益必须引起足够的重视。尽管已有多种DSP的检测方法,但这些检测方法各有其优缺点,至今尚未建立一种操作简便、快速而又可靠的常规检测方法。
本研究以HL-7702肝细胞为研究对象,使用OA标准品进行DSP细胞检测方法的研究。首先从细胞增殖、形态学变化及细胞凋亡等方面探讨OA的细胞毒性效应。然后使用激光共聚焦显微镜观察和检测OA对细胞F-肌动蛋白的影响,在此基础上,进一步利用TECAN Infiite M1000多功能酶标仪定量检测OA作用细胞后F-肌动蛋白解聚效应大小,初步建立可以用于检测DSP的细胞F-肌动蛋白检测法。使用细胞F-肌动蛋白检测法进行贝类样品的检测,并将检测结果与小鼠生物法及ELISA法的样品检测结果进行比较,以验证该方法的可行性。
本研究的主要结论如下:
(1) OA对HL-7702肝细胞增殖的抑制呈现时间、剂量-效应关系,染毒24 h的IC50为89.684 nmol/L。
(2) OA作用于HL-7702肝细胞24 h后,细胞形态发生明显变化,出现细胞凋亡形态学特征。使用Annexin V-FITC/PI双染流式细胞术进行细胞凋亡的检测,发现5 nmol/L OA即可诱导细胞发生凋亡,且细胞早期凋亡率随着剂量的增加而增加。
(3) OA作用于HL-7702肝细胞24 h可以使F-肌动蛋白发生解聚,且解聚率与OA浓度在2.5~40 nmol/L范围内具有线性关系。
(4)利用甲醇进行贝类样品的提取后需要进行氮气吹干,样品检测液中的甲醇浓度必须低于4%。
(5)细胞F-肌动蛋白检测法具有良好的特异性及重复性,基质效应低,检测限可达到2.5 nmol/L,相当于2.01μg/100 g。
(6)将ELISA法检测结果呈阳性的样品使用细胞F-肌动蛋白检测法进行检测,两种检测方法的检测结果具有较好的相关性。与ELISA法(检测限为10μg/100 g)相比,细胞F-肌动蛋白检测法具有更低的检测限,能更有效地保障贝类的食用安全。
Diarrhetic shellfish poisoning (DSP) are a group of marine toxins, which usually coexist with shellfish and consist primarily of okadaic acid (OA) and its derivatives. The most commonly reported symptom of human intoxication was diarrhoea. OA has been reported as a tumor promoter and it may pose a threat to human health even at extremely low concentration, so the long term effects of OA must be attentioned. A variety of methods with both advantages and disadvantages have been developed for the detection of DSP, but a convenient, rapid and accurate routine method is not yet developed.
HL-7702 Human liver cell line was employed to study the cell bioassay for DSP using OA standard solution. Firstly, the cytotoxic effect of OA on cell proliferation was investigated by cell counting kit-8, while the apoptosis induced by OA was examed by morphology and flow cytometry. Then, Images of HL-7702 was scanned by confocal laser scanning microscope after being treated with OA. Furthermore, TECAN Infiite M1000 was used for the quantitation of F-actin depolymerization and the cell F-actin assay was primarily developed for the detection of DSP. The feasibility of this assay was evaluated by comparative analysis of shellfish samples by the cell F-actin assay, mouse bioassay and ELISA. Main conclusions are as follow:
(1) OA inhibited the proliferation of HL-7702 significantly in a time and dose dependent manner with IC50 of OA being 89.684 nmol/L when the exposure time is 24 h.
(2) After 24 h of exposure, OA induced morphological changes in HL-7702 which consist with the characteristics of cell apoptosis. Then cell apoptosis was detected by flow cytometry after Annexin V-FITC and PI double staining. The result showed that OA can induce cell apoptosis when the concentration was as low as 5 nmol/L, and the early apoptotic ratio increased along with the increase of OA concentration.
(3) After 24 h of exposure, OA induced depolymerization of F-actin in HL-7702 significantly, showing a linear relationship with the concentration of OA in the range of 2.5~40 nmol/L.
(4) The methanolic extract obtained from shellfish sample should be dried under nitrogen, keeping the concentration of methanol below 4%.
(5) The cell F-actin assay showed a specific, reproducible and accurate detection for DSP, having a detection limit of 2.5 nmol/L which correspond to 2.01μg/100 g.
(6) 42 samples, previously detected positive by ELISA, were also submitted to the cell F-actin assay. The results obtained by cell F-actin assay correlated well with those obtained by ELISA. Compared with ELISA, the cell F-actin assay had lower detection limit, thus could assure the safety of shellfish more effectively.
本研究以HL-7702肝细胞为研究对象,使用OA标准品进行DSP细胞检测方法的研究。首先从细胞增殖、形态学变化及细胞凋亡等方面探讨OA的细胞毒性效应。然后使用激光共聚焦显微镜观察和检测OA对细胞F-肌动蛋白的影响,在此基础上,进一步利用TECAN Infiite M1000多功能酶标仪定量检测OA作用细胞后F-肌动蛋白解聚效应大小,初步建立可以用于检测DSP的细胞F-肌动蛋白检测法。使用细胞F-肌动蛋白检测法进行贝类样品的检测,并将检测结果与小鼠生物法及ELISA法的样品检测结果进行比较,以验证该方法的可行性。
本研究的主要结论如下:
(1) OA对HL-7702肝细胞增殖的抑制呈现时间、剂量-效应关系,染毒24 h的IC50为89.684 nmol/L。
(2) OA作用于HL-7702肝细胞24 h后,细胞形态发生明显变化,出现细胞凋亡形态学特征。使用Annexin V-FITC/PI双染流式细胞术进行细胞凋亡的检测,发现5 nmol/L OA即可诱导细胞发生凋亡,且细胞早期凋亡率随着剂量的增加而增加。
(3) OA作用于HL-7702肝细胞24 h可以使F-肌动蛋白发生解聚,且解聚率与OA浓度在2.5~40 nmol/L范围内具有线性关系。
(4)利用甲醇进行贝类样品的提取后需要进行氮气吹干,样品检测液中的甲醇浓度必须低于4%。
(5)细胞F-肌动蛋白检测法具有良好的特异性及重复性,基质效应低,检测限可达到2.5 nmol/L,相当于2.01μg/100 g。
(6)将ELISA法检测结果呈阳性的样品使用细胞F-肌动蛋白检测法进行检测,两种检测方法的检测结果具有较好的相关性。与ELISA法(检测限为10μg/100 g)相比,细胞F-肌动蛋白检测法具有更低的检测限,能更有效地保障贝类的食用安全。
Diarrhetic shellfish poisoning (DSP) are a group of marine toxins, which usually coexist with shellfish and consist primarily of okadaic acid (OA) and its derivatives. The most commonly reported symptom of human intoxication was diarrhoea. OA has been reported as a tumor promoter and it may pose a threat to human health even at extremely low concentration, so the long term effects of OA must be attentioned. A variety of methods with both advantages and disadvantages have been developed for the detection of DSP, but a convenient, rapid and accurate routine method is not yet developed.
HL-7702 Human liver cell line was employed to study the cell bioassay for DSP using OA standard solution. Firstly, the cytotoxic effect of OA on cell proliferation was investigated by cell counting kit-8, while the apoptosis induced by OA was examed by morphology and flow cytometry. Then, Images of HL-7702 was scanned by confocal laser scanning microscope after being treated with OA. Furthermore, TECAN Infiite M1000 was used for the quantitation of F-actin depolymerization and the cell F-actin assay was primarily developed for the detection of DSP. The feasibility of this assay was evaluated by comparative analysis of shellfish samples by the cell F-actin assay, mouse bioassay and ELISA. Main conclusions are as follow:
(1) OA inhibited the proliferation of HL-7702 significantly in a time and dose dependent manner with IC50 of OA being 89.684 nmol/L when the exposure time is 24 h.
(2) After 24 h of exposure, OA induced morphological changes in HL-7702 which consist with the characteristics of cell apoptosis. Then cell apoptosis was detected by flow cytometry after Annexin V-FITC and PI double staining. The result showed that OA can induce cell apoptosis when the concentration was as low as 5 nmol/L, and the early apoptotic ratio increased along with the increase of OA concentration.
(3) After 24 h of exposure, OA induced depolymerization of F-actin in HL-7702 significantly, showing a linear relationship with the concentration of OA in the range of 2.5~40 nmol/L.
(4) The methanolic extract obtained from shellfish sample should be dried under nitrogen, keeping the concentration of methanol below 4%.
(5) The cell F-actin assay showed a specific, reproducible and accurate detection for DSP, having a detection limit of 2.5 nmol/L which correspond to 2.01μg/100 g.
(6) 42 samples, previously detected positive by ELISA, were also submitted to the cell F-actin assay. The results obtained by cell F-actin assay correlated well with those obtained by ELISA. Compared with ELISA, the cell F-actin assay had lower detection limit, thus could assure the safety of shellfish more effectively.
引文
[1]陈杰,李春盛,曹雅明,等.赤潮藻类毒素检测方法的研究概况[J].中华预防医学杂志,2001,35(2):130-132
[2]马敬军,曾名勇,周德庆.贝类毒素检验及排除方法的研究进展[J].中国海洋药物,2003,22(6):41-45
[3]李淑冰,李惠珍,许旭萍.贝毒素的研究现状及产生源探究[J].食品科学,2000,21(5):39-41
[4]余亚英,袁唯.贝类毒素及检测方法的研究进展[J].食品研究与开发,2007,28(10):175-178
[5]杨莉,杨维东,刘洁生,等.广州市售贝类麻痹性贝毒和腹泻性贝毒污染状况分析[J].卫生研究,2006,35(4):435-439
[6] Azanza RU, Taylor FJRM. Are Pyrodinium blooms in the Southeast Asian region recurring and spreading ? A view at the end of the millennium[J]. Ambio, 2001, 30(6): 356-364
[7] Torgersen T, Aasen J, Aune T. Diarrhetic shellfish poisoning by okadaic acid esters from Brown crabs(Cancer pagurus) in Norway[J]. Toxicon, 2005, 46(5):572-578
[8]孙迎春,陈杰,刘军,等.抗软海绵酸单克隆抗体的制备[J].中国公共生,2004,20(2):186-188
[9]邬建勇,王金辉,程祥圣.上海市海产品市场的麻痹性贝毒检测和分析[J].毒理学杂志,2006,20(1):25-26
[10] GB/T 5009.212,贝类中腹泻性贝类毒素的测定[S].北京:中国标准出版社,2008
[11] Yasumoto T, Oshima Y, Yamaguchi M. Occurrence of a new type of shellfish poisoning in the Tohoku District[J]. Bull Jpn Soc Sci Fish, 1978, 44(11):1249-1255
[12]丁君.赤潮毒素中腹泻性贝毒和麻痹性贝毒的研究及进展[J].大连水产学院学报,2001,16(3):212-218
[13]陈则玲,付云娜,巩宁.腹泻性贝毒及其高效液相色谱检测方法[J].海洋通报,2000,19(1):73-78
[14]陈洋,颜天,于仁成,等.OA对人肝细胞HL-7702和肝癌细胞Bel-7402增殖的影响和对细胞凋亡的诱导作用研究[J].中山大学学报,2008,47(5):86-92
[15]杨维东,彭喜春,刘洁生,等.腹泻性贝毒研究现状[J].海洋科学,2005,29(5):66-72
[16]卢士英.海产品中大田软海绵酸快速检测方法研究[D].吉林:吉林大学,2007
[17] Zhou M, Li J, Luckas B, et al. A recent shellfish toxin investigation in China[J]. Mar Pollut Bull, 1999, 39:331-334
[18]吴施卫,张纯超,卢楚谦,等.南海近岸海域腹泻性贝类毒素分析[J].海洋环境科学,2005,24(4):48-51
[19]赵晓芳,计融,腹泻性贝类毒素研究进展[J].中国热带医学,2006,6(l):157-159
[20] Burgess V, Shaw G. Pectenotoxins-an issue for public health: a review of their comparative toxicology and metabolism[J]. Environ Int, 2001, 27(4):275-283
[21]雷芳.深圳市售贝类麻痹性贝类毒素和腹泻性贝类毒素污染状况研究[D].广州:暨南大学,2009
[22] Mouratidou T, Kaniou-Grigoriadou I, Samara C, et al. Detection of the marine toxin okadaic acid in mussels during a diarrhetic shellfish poisoning (DSP) episode in Thermaikos Gulf, Greece, using biological, chemical and immunological methods[J]. Sci Total Environ, 2006, 366(2-3):894-904
[23] Daranas AH, Norte M, Fernández JJ. Toxic marine microalgae[J]. Toxicon, 2001, 39(8):1101-1132
[24] Campàs M, Prieto-Simón B, Marty JL. Biosensors to detect marine toxins: assessing seafood safety[J]. Talanta, 2007, 72(3): 884-895
[25] Li DW, Liu JP, Schmid PC, et al. Protein serine/threonine phosphatase-1 dephosphorylates p53 at Ser-15 and Ser-37 to modulate its transcriptional and apoptotic activities[J]. Oncogene, 2006, 25(21):3006-3022
[26] Creppy EE, TraoréA, Baudrimont I, et al. Recent advances in the study of epigenetic effects induced by the phycotoxin okadaic acid[J]. Toxicology, 2002, 181-182:433-439
[27] Casarini L, Franchini A, Malagoli D, et al. Evaluation of the effects of the marine toxin okadaic acid by using FETAX assay[J]. Toxicol Lett, 2007, 169(2):145-151
[28] TraoréA, Ruiz S, Baudrimont I, et al. Combined effects of okadaic acid and cadmium on lipid peroxidation and DNA bases modifications (m5dC and 8-(OH)-dG) in Caco-2 cells[J]. Arch Toxicol, 2000, 74(2):79-84
[29] Montilla-López P, Mu?oz-Agueda MC, Feijóo López M, et al. Comparison of melatonin versus vitamin C on oxidative stress and antioxidant enzyme activity in Alzheimer's disease induced by okadaic acid in neuroblastoma cells[J]. Eur J Pharmacol, 2002, 451(3):237-243
[30] Lago J, Santaclara F, Vieites JM, et al. Collapse of mitochondrial membrane potential and caspases activation are early events in okadaic acid-treated Caco-2 cells[J]. Toxicon, 2005, 46(5):579-586
[31] Berven G, Saetre F, Halvorsen K, et al. Effects of the diarrhetic shellfish toxin, okadaic acid, on cytoskeletal elements, viability and functionality of rat liver and intestinal cells[J]. Toxicon, 2001, 39(2-3):349-362
[32] Souid-Mensi G, Moukha S, Mobio TA, et al. The cytotoxicity and genotoxicity of okadaic acid are cell-line dependent[J]. Toxicon, 2008, 51(8):1338-1344
[33] Matias WG, Traore A, Bonini M, et al. Oxygen reactive radicals production in cell culture by okadaic acid and their implication in protein synthesis inhibition[J]. Hum Exp Toxicol, 1999, 18(10):634-639
[34] Koike K, Sato S, Yamaji M, et al. Occurrence of okadaic acid-producing Prorocentrum lima on the Sanriku coast, northern Japan[J]. Toxicon, 1998, 36(12):2039-2042
[35] Toyofuku H. Joint FAO/WHO/IOC activities to provide scientific advice on marine biotoxins (research report)[J]. Mar Pollut Bull, 2006, 52(12):1735-1745
[36] Camacho FG, Rodríguez JG, Mirón AS, et al. Biotechnological significance of toxic marine dinoflagellates[J]. Biotechnol Adv, 2007, 25(2):176-194
[37] Rundberget T, Gustad E, Samdal IA, et al. A convenient and cost-effective method for monitoring marine algal toxins with passive samplers[J]. Toxicon, 2009, 53(5):543-550
[38] Vale P, Sampayo MA. First confirmation of human diarrhoeic poisonings by okadaic acid esters after ingestion of razor clams (Solen marginatus) and green crabs (Carcinus maenas) in Aveiro lagoon, Portugal and detection of okadaic acid esters in phytoplankton[J]. Toxicon, 2002, 40(7):989-996
[39] Torgersen T, Bremnes NB, Rundberget T, et al. Structural confirmation and occurrence of azaspiracids in Scandinavian brown crabs (Cancer pagurus)[J]. Toxicon, 2008, 51(1):93-101
[40] Prassopoulou E, Katikou P, Georgantelis D, et al. Detection of okadaic acid and related esters in mussels during diarrhetic shellfish poisoning (DSP) episodes in Greece using the mouse bioassay, the PP2A inhibition assay and HPLC with fluorimetric detection[J]. Toxicon, 2009, 53(2):214-227
[41] Ciminiello P, Fattorusso E. Bivalve molluscs as vectors of marine biotoxins involved in seafood poisoning[J]. Prog Mol Subcell Biol, 2006, 43:53-82
[42] Swaddiwudhipong W, Kunasol P, Sangwanloy O, et al. Foodborne disease outbreaks of chemical etiology in Thailand, 1981-1987[J]. Southeast Asian J Trop Med Public Health, 1999, 20(1):125-132
[43] Mak KC, Yu H, Choi MC, et al. Okadaic acid, a causative toxin of diarrhetic shellfish poisoning, in green-lipped mussels Perna viridis from Hong Kong fish culture zones: method development and monitoring[J]. Mar Pollut Bull, 2005, 51(8-12):1010-1017
[44]周名江,李钧,于仁诚.赤潮藻毒素研究进展[J].中国海洋药物,1999,18(3):48-54
[45]刘仁沿.我国贝类中赤潮毒素腹泻性贝毒免疫检测技术研究[D].大连:大连海事大学,2008
[46]黄爱君,黄海燕,刘建军.麻痹性和腹泻性贝类毒素的检测方法研究进展[J].环境与健康杂志,2010,27(1):84-86
[47]江天久,尹伊伟,骆育敏,等.大亚湾麻痹性贝类毒素HPLC分析[J].海洋环境科学,2000,19(3):16-19
[48] Li A, Yu R, Li J, et al. Protein phosphatase inhibition assay for detection of diarrhetic shellfish poison in oyster[J]. Chin J Anal Chem, 2006, 34(3):283-287
[49] Lee JS, Yanagi T, Kenma R, et al. Fluorometric determination of diarrhetic shell?sh toxins by high-performance liquid chromatography[J]. Agric Biol Chem, 1987, 51:877-881
[50]曹际娟,卫锋,马惠蕊,等.贝类毒素检测技术及研究进展[J].检验检疫科学,2004,14(1):53-56
[51]傅云娜,陈则玲.腹泻性贝毒的高效液相色谱法测定条件改进及其运用[J].海洋通报,2003,22(1):92-95
[52]陈晓红,张卫红,张倩芝.液相色谱和液质联用技术在水产品安全检测中的应用[J].广州化工,2005,33(3):16-19
[53] Paz B, Daranas AH, Norte M, et al. Yessotoxins, a group of marine polyether toxins: an overview[J]. Mar Drugs, 2008, 6(2):73-102
[54] Christian B, Luckas B. Determination of marine biotoxins relevant for regulations: from the mouse bioassay to coupled LC-MS methods[J]. Anal Bioanal Chem, 2008, 391(1):117-134
[55]于兵,曹际娟,尤永莉,等.ELISA与小白鼠生物法检测贝类中麻痹性贝毒的比较[J].检疫检疫科学,2005,15(1):32-35
[56] Levine L, Fujiki H, Yamada K, et al. Production of antibodies and development of a radioimmunoassay for okadaic acid[J]. Toxicon, 1988, 26(12):1123-1128
[57] Kreuzer MP, Pravda M, O'Sullivan CK, et al. Novel electrochemical immunosensors for seafood toxin analysis[J]. Toxicon, 2002, 40(9):1267-1274
[58] Tsafack VC, Marquette CA, Leca B, et al. An electrochemiluminescence-based fibre optic biosensor for choline flow injection analysis[J]. Analyst, 2000, 125(1):151-155
[59] Campàs M, Marty JL. Enzyme sensor for the electrochemical detection of the marine toxin okadaic acid[J]. Anal Chem Acta, 2007, 605(1):87-93
[60] Llamas NM, Stewart L, Fodey T, et al. Development of a novel immunobiosensor method for the rapid detection of okadaic acid contamination in shellfish extracts[J]. Anal Chem Acta, 2007, 389(2):581-587
[61] Nú?ez PE, Scoging AC. Comparison of a protein phosphatase inhibition assay, HPLC assay and enzyme-linked immunosorbent assay with the mouse bioassay forthe detection of diarrhetic shellfish poisoning toxins in European shellfish[J]. Int J Food Microbiol, 1997, 36(1):39-48
[62] Vieytes MR, Fontal OI, Leira F, et al. A ?uorescent microplate assay for diarrheic shell?sh toxins[J]. Anal Biochem, 1997, 248(2):258-264
[63] Mountfort DO, Kennedy G, Garthwaite I, et al. Evaluation of ?uorometric protein phosphatase inhibition assay in the determination of okadaic acid in mussels[J]. Toxicon, 1999, 37(6):909-922
[64] Albano C, Ronzitti G, Rossini AM, et al. The total activity of a mixture of okadaic acid-group compounds can be calculated by those of individual analogues in a phosphoprotein phosphatase 2A assay[J]. Toxicon, 2009, 53(6):631-637
[65] Rossini GP. Functional assays in marine biotoxin detection[J]. Toxicology, 2005, 207(3):451-462
[66] Ca?ete E, Diogène J. Comparative study of the use of neuroblastoma cells (Neuro-2a) and neuroblastomaxglioma hybrid cells (NG108-15) for the toxic effect quantification of marine toxins[J]. Toxicon, 2008, 52(4):541-550
[67] Hayashi R, Saito H, Okumura M, et al. Cell bioassay for paralytic shellfish poisoning (PSP): comparison with postcolumn derivatization liquid chromatographic analysis and application to the monitoring of PSP in shellfish[J]. J Agric Food Chem, 2006 54(2):269-273
[68] Caillaud A, Ca?ete E, de la Iglesia P, et al. Cell-based assay coupled with chromatographic fractioning: a strategy for marine toxins detection in natural samples[J]. Toxicol In Vitro, 2009, 23(8):1591-1596
[69] Leira F, Vieites JM, Vieytes MR, et al. Apoptotic events induced by the phosphatase inhibitor okadaic acid in normal human lung fibroblasts[J]. Toxicol In Vitro, 2001, 15(3):199-208
[70] Flanagan AF, Callanan KR, Donlon J, et al. A cytotoxicity assay for the detection and differentiation of two families of shellfish toxins[J]. Toxicon, 2001, 39(7):1021-1027
[71] Fessard V, Diogène G, Dubreuil A, et al. Selection of cytotoxic responses to maitotoxin and okadaic acid and evaluation of toxicity of dinoflagellate extracts[J].Nat Toxins, 1994, 2(5):322-328
[72] Korsnes MS, Hetland DL, Espenes A, et al. Apoptotic events induced by yessotoxin in myoblast cell lines from rat and mouse[J]. Toxicol In Vitro, 2006, 20(7):1077-1087
[73] Leira F, Alvarez C, Vieites JM, et al. Study of cytoskeletal changes induced by okadaic acid in BE(2)-M17 cells by means of a quantitative fluorimetric microplate assay[J]. Toxicol In Vitro, 2001, 15(4-5):277-282
[74] Leira F, Cabado AG, Vieytes MR, et al. Characterization of F-actin depolymerization as a major toxic event induced by pectenotoxin-6 in neuroblastoma cells[J]. Biochem Pharmacol, 2002, 63(11):1979-1988
[75] Aune T, Yasumoto T, Engeland E. Light and scanning electron microscopic studies on effects of marine algal toxins toward freshly prepared hepatocytes[J]. Toxicol Environ Health, 1991, 34(1):1-9
[76] Tubaro A, Florio C, Luxich E, et al. Suitability of the MTT-based cytotoxicity assay to detect okadaic acid contamination of mussels[J]. Toxicon, 1996, 34(9):965-974
[77] Leira F, Alvarez C, Cabado AG, et al. Development of a F actin-based live-cell fluorimetric microplate assay for diarrhetic shellfish toxins[J]. Anal Biochem, 2003, 17(2):129-135
[78]周名江,朱明远,张经.中国赤潮的发生趋势和研究进展[J].生命科学,2001,13(2):54-59
[79] Jayaraj R, Gupta N, Rao PV. Multiple signal transduction pathways in okadaic acid induced apoptosis in HeLa cells[J]. Toxicology, 2009, 256(1):118-127
[80]陈谋通.三氯乙烯致L-02肝细胞毒性后SET与其它蛋白质相互作用研究[D].广州:华南理工大学,2009
[81] Evan G, Littlewood T. A matter of life and cell death[J]. Science, 1998, 281(5381): 1317-1322
[82]陈洋. DSP等赤潮藻毒素对哺乳类细胞的毒性效应及机制研究[D].青岛:中国科学院海洋研究所,2008
[83] Klumpp S, Krieglstein J. Serine/threonine protein phosphatases in apoptosis[J].Curr Opin Pharmacol, 2002, 2(4) : 458-462
[84] Fernández JJ, Candenas ML, Souto ML, et al. Okadaic acid, useful tool for studying cellular processes[J]. Curr Med Chem. 2002, 9(2):229-262
[85] Rando OJ, Zhao K, Crabtree GR. Searching for a function for nuclear actin[J]. Trends Cell Biol, 2000, 10(3):92-97
[86]朱筱娟,曾宪录,宋朝霞,等.细胞核内肌动蛋白及其功能研究进展[J].科学通报,2004,49(11):1031-1035
[87] GenescàM, Sola A, Hotter G. Actin cytoskeleton derangement induces apoptosis in renal ischemia/reperfusion[J]. Apoptosis, 2006, 11(4):563-571
[88] Botana LM, Alfonso A, Botana A, et al. Functional assays for marine toxins as an alternative, high-throughput-screening solution to animal tests[J]. Trends in Analytical Chemistry, 2009, 28(5):603-611
[89]余新天,黄海燕,付英斌,等.贝类毒素昆明种小鼠生物测定法建立[J].中国公共卫生,2009,25(5):590-591
[2]马敬军,曾名勇,周德庆.贝类毒素检验及排除方法的研究进展[J].中国海洋药物,2003,22(6):41-45
[3]李淑冰,李惠珍,许旭萍.贝毒素的研究现状及产生源探究[J].食品科学,2000,21(5):39-41
[4]余亚英,袁唯.贝类毒素及检测方法的研究进展[J].食品研究与开发,2007,28(10):175-178
[5]杨莉,杨维东,刘洁生,等.广州市售贝类麻痹性贝毒和腹泻性贝毒污染状况分析[J].卫生研究,2006,35(4):435-439
[6] Azanza RU, Taylor FJRM. Are Pyrodinium blooms in the Southeast Asian region recurring and spreading ? A view at the end of the millennium[J]. Ambio, 2001, 30(6): 356-364
[7] Torgersen T, Aasen J, Aune T. Diarrhetic shellfish poisoning by okadaic acid esters from Brown crabs(Cancer pagurus) in Norway[J]. Toxicon, 2005, 46(5):572-578
[8]孙迎春,陈杰,刘军,等.抗软海绵酸单克隆抗体的制备[J].中国公共生,2004,20(2):186-188
[9]邬建勇,王金辉,程祥圣.上海市海产品市场的麻痹性贝毒检测和分析[J].毒理学杂志,2006,20(1):25-26
[10] GB/T 5009.212,贝类中腹泻性贝类毒素的测定[S].北京:中国标准出版社,2008
[11] Yasumoto T, Oshima Y, Yamaguchi M. Occurrence of a new type of shellfish poisoning in the Tohoku District[J]. Bull Jpn Soc Sci Fish, 1978, 44(11):1249-1255
[12]丁君.赤潮毒素中腹泻性贝毒和麻痹性贝毒的研究及进展[J].大连水产学院学报,2001,16(3):212-218
[13]陈则玲,付云娜,巩宁.腹泻性贝毒及其高效液相色谱检测方法[J].海洋通报,2000,19(1):73-78
[14]陈洋,颜天,于仁成,等.OA对人肝细胞HL-7702和肝癌细胞Bel-7402增殖的影响和对细胞凋亡的诱导作用研究[J].中山大学学报,2008,47(5):86-92
[15]杨维东,彭喜春,刘洁生,等.腹泻性贝毒研究现状[J].海洋科学,2005,29(5):66-72
[16]卢士英.海产品中大田软海绵酸快速检测方法研究[D].吉林:吉林大学,2007
[17] Zhou M, Li J, Luckas B, et al. A recent shellfish toxin investigation in China[J]. Mar Pollut Bull, 1999, 39:331-334
[18]吴施卫,张纯超,卢楚谦,等.南海近岸海域腹泻性贝类毒素分析[J].海洋环境科学,2005,24(4):48-51
[19]赵晓芳,计融,腹泻性贝类毒素研究进展[J].中国热带医学,2006,6(l):157-159
[20] Burgess V, Shaw G. Pectenotoxins-an issue for public health: a review of their comparative toxicology and metabolism[J]. Environ Int, 2001, 27(4):275-283
[21]雷芳.深圳市售贝类麻痹性贝类毒素和腹泻性贝类毒素污染状况研究[D].广州:暨南大学,2009
[22] Mouratidou T, Kaniou-Grigoriadou I, Samara C, et al. Detection of the marine toxin okadaic acid in mussels during a diarrhetic shellfish poisoning (DSP) episode in Thermaikos Gulf, Greece, using biological, chemical and immunological methods[J]. Sci Total Environ, 2006, 366(2-3):894-904
[23] Daranas AH, Norte M, Fernández JJ. Toxic marine microalgae[J]. Toxicon, 2001, 39(8):1101-1132
[24] Campàs M, Prieto-Simón B, Marty JL. Biosensors to detect marine toxins: assessing seafood safety[J]. Talanta, 2007, 72(3): 884-895
[25] Li DW, Liu JP, Schmid PC, et al. Protein serine/threonine phosphatase-1 dephosphorylates p53 at Ser-15 and Ser-37 to modulate its transcriptional and apoptotic activities[J]. Oncogene, 2006, 25(21):3006-3022
[26] Creppy EE, TraoréA, Baudrimont I, et al. Recent advances in the study of epigenetic effects induced by the phycotoxin okadaic acid[J]. Toxicology, 2002, 181-182:433-439
[27] Casarini L, Franchini A, Malagoli D, et al. Evaluation of the effects of the marine toxin okadaic acid by using FETAX assay[J]. Toxicol Lett, 2007, 169(2):145-151
[28] TraoréA, Ruiz S, Baudrimont I, et al. Combined effects of okadaic acid and cadmium on lipid peroxidation and DNA bases modifications (m5dC and 8-(OH)-dG) in Caco-2 cells[J]. Arch Toxicol, 2000, 74(2):79-84
[29] Montilla-López P, Mu?oz-Agueda MC, Feijóo López M, et al. Comparison of melatonin versus vitamin C on oxidative stress and antioxidant enzyme activity in Alzheimer's disease induced by okadaic acid in neuroblastoma cells[J]. Eur J Pharmacol, 2002, 451(3):237-243
[30] Lago J, Santaclara F, Vieites JM, et al. Collapse of mitochondrial membrane potential and caspases activation are early events in okadaic acid-treated Caco-2 cells[J]. Toxicon, 2005, 46(5):579-586
[31] Berven G, Saetre F, Halvorsen K, et al. Effects of the diarrhetic shellfish toxin, okadaic acid, on cytoskeletal elements, viability and functionality of rat liver and intestinal cells[J]. Toxicon, 2001, 39(2-3):349-362
[32] Souid-Mensi G, Moukha S, Mobio TA, et al. The cytotoxicity and genotoxicity of okadaic acid are cell-line dependent[J]. Toxicon, 2008, 51(8):1338-1344
[33] Matias WG, Traore A, Bonini M, et al. Oxygen reactive radicals production in cell culture by okadaic acid and their implication in protein synthesis inhibition[J]. Hum Exp Toxicol, 1999, 18(10):634-639
[34] Koike K, Sato S, Yamaji M, et al. Occurrence of okadaic acid-producing Prorocentrum lima on the Sanriku coast, northern Japan[J]. Toxicon, 1998, 36(12):2039-2042
[35] Toyofuku H. Joint FAO/WHO/IOC activities to provide scientific advice on marine biotoxins (research report)[J]. Mar Pollut Bull, 2006, 52(12):1735-1745
[36] Camacho FG, Rodríguez JG, Mirón AS, et al. Biotechnological significance of toxic marine dinoflagellates[J]. Biotechnol Adv, 2007, 25(2):176-194
[37] Rundberget T, Gustad E, Samdal IA, et al. A convenient and cost-effective method for monitoring marine algal toxins with passive samplers[J]. Toxicon, 2009, 53(5):543-550
[38] Vale P, Sampayo MA. First confirmation of human diarrhoeic poisonings by okadaic acid esters after ingestion of razor clams (Solen marginatus) and green crabs (Carcinus maenas) in Aveiro lagoon, Portugal and detection of okadaic acid esters in phytoplankton[J]. Toxicon, 2002, 40(7):989-996
[39] Torgersen T, Bremnes NB, Rundberget T, et al. Structural confirmation and occurrence of azaspiracids in Scandinavian brown crabs (Cancer pagurus)[J]. Toxicon, 2008, 51(1):93-101
[40] Prassopoulou E, Katikou P, Georgantelis D, et al. Detection of okadaic acid and related esters in mussels during diarrhetic shellfish poisoning (DSP) episodes in Greece using the mouse bioassay, the PP2A inhibition assay and HPLC with fluorimetric detection[J]. Toxicon, 2009, 53(2):214-227
[41] Ciminiello P, Fattorusso E. Bivalve molluscs as vectors of marine biotoxins involved in seafood poisoning[J]. Prog Mol Subcell Biol, 2006, 43:53-82
[42] Swaddiwudhipong W, Kunasol P, Sangwanloy O, et al. Foodborne disease outbreaks of chemical etiology in Thailand, 1981-1987[J]. Southeast Asian J Trop Med Public Health, 1999, 20(1):125-132
[43] Mak KC, Yu H, Choi MC, et al. Okadaic acid, a causative toxin of diarrhetic shellfish poisoning, in green-lipped mussels Perna viridis from Hong Kong fish culture zones: method development and monitoring[J]. Mar Pollut Bull, 2005, 51(8-12):1010-1017
[44]周名江,李钧,于仁诚.赤潮藻毒素研究进展[J].中国海洋药物,1999,18(3):48-54
[45]刘仁沿.我国贝类中赤潮毒素腹泻性贝毒免疫检测技术研究[D].大连:大连海事大学,2008
[46]黄爱君,黄海燕,刘建军.麻痹性和腹泻性贝类毒素的检测方法研究进展[J].环境与健康杂志,2010,27(1):84-86
[47]江天久,尹伊伟,骆育敏,等.大亚湾麻痹性贝类毒素HPLC分析[J].海洋环境科学,2000,19(3):16-19
[48] Li A, Yu R, Li J, et al. Protein phosphatase inhibition assay for detection of diarrhetic shellfish poison in oyster[J]. Chin J Anal Chem, 2006, 34(3):283-287
[49] Lee JS, Yanagi T, Kenma R, et al. Fluorometric determination of diarrhetic shell?sh toxins by high-performance liquid chromatography[J]. Agric Biol Chem, 1987, 51:877-881
[50]曹际娟,卫锋,马惠蕊,等.贝类毒素检测技术及研究进展[J].检验检疫科学,2004,14(1):53-56
[51]傅云娜,陈则玲.腹泻性贝毒的高效液相色谱法测定条件改进及其运用[J].海洋通报,2003,22(1):92-95
[52]陈晓红,张卫红,张倩芝.液相色谱和液质联用技术在水产品安全检测中的应用[J].广州化工,2005,33(3):16-19
[53] Paz B, Daranas AH, Norte M, et al. Yessotoxins, a group of marine polyether toxins: an overview[J]. Mar Drugs, 2008, 6(2):73-102
[54] Christian B, Luckas B. Determination of marine biotoxins relevant for regulations: from the mouse bioassay to coupled LC-MS methods[J]. Anal Bioanal Chem, 2008, 391(1):117-134
[55]于兵,曹际娟,尤永莉,等.ELISA与小白鼠生物法检测贝类中麻痹性贝毒的比较[J].检疫检疫科学,2005,15(1):32-35
[56] Levine L, Fujiki H, Yamada K, et al. Production of antibodies and development of a radioimmunoassay for okadaic acid[J]. Toxicon, 1988, 26(12):1123-1128
[57] Kreuzer MP, Pravda M, O'Sullivan CK, et al. Novel electrochemical immunosensors for seafood toxin analysis[J]. Toxicon, 2002, 40(9):1267-1274
[58] Tsafack VC, Marquette CA, Leca B, et al. An electrochemiluminescence-based fibre optic biosensor for choline flow injection analysis[J]. Analyst, 2000, 125(1):151-155
[59] Campàs M, Marty JL. Enzyme sensor for the electrochemical detection of the marine toxin okadaic acid[J]. Anal Chem Acta, 2007, 605(1):87-93
[60] Llamas NM, Stewart L, Fodey T, et al. Development of a novel immunobiosensor method for the rapid detection of okadaic acid contamination in shellfish extracts[J]. Anal Chem Acta, 2007, 389(2):581-587
[61] Nú?ez PE, Scoging AC. Comparison of a protein phosphatase inhibition assay, HPLC assay and enzyme-linked immunosorbent assay with the mouse bioassay forthe detection of diarrhetic shellfish poisoning toxins in European shellfish[J]. Int J Food Microbiol, 1997, 36(1):39-48
[62] Vieytes MR, Fontal OI, Leira F, et al. A ?uorescent microplate assay for diarrheic shell?sh toxins[J]. Anal Biochem, 1997, 248(2):258-264
[63] Mountfort DO, Kennedy G, Garthwaite I, et al. Evaluation of ?uorometric protein phosphatase inhibition assay in the determination of okadaic acid in mussels[J]. Toxicon, 1999, 37(6):909-922
[64] Albano C, Ronzitti G, Rossini AM, et al. The total activity of a mixture of okadaic acid-group compounds can be calculated by those of individual analogues in a phosphoprotein phosphatase 2A assay[J]. Toxicon, 2009, 53(6):631-637
[65] Rossini GP. Functional assays in marine biotoxin detection[J]. Toxicology, 2005, 207(3):451-462
[66] Ca?ete E, Diogène J. Comparative study of the use of neuroblastoma cells (Neuro-2a) and neuroblastomaxglioma hybrid cells (NG108-15) for the toxic effect quantification of marine toxins[J]. Toxicon, 2008, 52(4):541-550
[67] Hayashi R, Saito H, Okumura M, et al. Cell bioassay for paralytic shellfish poisoning (PSP): comparison with postcolumn derivatization liquid chromatographic analysis and application to the monitoring of PSP in shellfish[J]. J Agric Food Chem, 2006 54(2):269-273
[68] Caillaud A, Ca?ete E, de la Iglesia P, et al. Cell-based assay coupled with chromatographic fractioning: a strategy for marine toxins detection in natural samples[J]. Toxicol In Vitro, 2009, 23(8):1591-1596
[69] Leira F, Vieites JM, Vieytes MR, et al. Apoptotic events induced by the phosphatase inhibitor okadaic acid in normal human lung fibroblasts[J]. Toxicol In Vitro, 2001, 15(3):199-208
[70] Flanagan AF, Callanan KR, Donlon J, et al. A cytotoxicity assay for the detection and differentiation of two families of shellfish toxins[J]. Toxicon, 2001, 39(7):1021-1027
[71] Fessard V, Diogène G, Dubreuil A, et al. Selection of cytotoxic responses to maitotoxin and okadaic acid and evaluation of toxicity of dinoflagellate extracts[J].Nat Toxins, 1994, 2(5):322-328
[72] Korsnes MS, Hetland DL, Espenes A, et al. Apoptotic events induced by yessotoxin in myoblast cell lines from rat and mouse[J]. Toxicol In Vitro, 2006, 20(7):1077-1087
[73] Leira F, Alvarez C, Vieites JM, et al. Study of cytoskeletal changes induced by okadaic acid in BE(2)-M17 cells by means of a quantitative fluorimetric microplate assay[J]. Toxicol In Vitro, 2001, 15(4-5):277-282
[74] Leira F, Cabado AG, Vieytes MR, et al. Characterization of F-actin depolymerization as a major toxic event induced by pectenotoxin-6 in neuroblastoma cells[J]. Biochem Pharmacol, 2002, 63(11):1979-1988
[75] Aune T, Yasumoto T, Engeland E. Light and scanning electron microscopic studies on effects of marine algal toxins toward freshly prepared hepatocytes[J]. Toxicol Environ Health, 1991, 34(1):1-9
[76] Tubaro A, Florio C, Luxich E, et al. Suitability of the MTT-based cytotoxicity assay to detect okadaic acid contamination of mussels[J]. Toxicon, 1996, 34(9):965-974
[77] Leira F, Alvarez C, Cabado AG, et al. Development of a F actin-based live-cell fluorimetric microplate assay for diarrhetic shellfish toxins[J]. Anal Biochem, 2003, 17(2):129-135
[78]周名江,朱明远,张经.中国赤潮的发生趋势和研究进展[J].生命科学,2001,13(2):54-59
[79] Jayaraj R, Gupta N, Rao PV. Multiple signal transduction pathways in okadaic acid induced apoptosis in HeLa cells[J]. Toxicology, 2009, 256(1):118-127
[80]陈谋通.三氯乙烯致L-02肝细胞毒性后SET与其它蛋白质相互作用研究[D].广州:华南理工大学,2009
[81] Evan G, Littlewood T. A matter of life and cell death[J]. Science, 1998, 281(5381): 1317-1322
[82]陈洋. DSP等赤潮藻毒素对哺乳类细胞的毒性效应及机制研究[D].青岛:中国科学院海洋研究所,2008
[83] Klumpp S, Krieglstein J. Serine/threonine protein phosphatases in apoptosis[J].Curr Opin Pharmacol, 2002, 2(4) : 458-462
[84] Fernández JJ, Candenas ML, Souto ML, et al. Okadaic acid, useful tool for studying cellular processes[J]. Curr Med Chem. 2002, 9(2):229-262
[85] Rando OJ, Zhao K, Crabtree GR. Searching for a function for nuclear actin[J]. Trends Cell Biol, 2000, 10(3):92-97
[86]朱筱娟,曾宪录,宋朝霞,等.细胞核内肌动蛋白及其功能研究进展[J].科学通报,2004,49(11):1031-1035
[87] GenescàM, Sola A, Hotter G. Actin cytoskeleton derangement induces apoptosis in renal ischemia/reperfusion[J]. Apoptosis, 2006, 11(4):563-571
[88] Botana LM, Alfonso A, Botana A, et al. Functional assays for marine toxins as an alternative, high-throughput-screening solution to animal tests[J]. Trends in Analytical Chemistry, 2009, 28(5):603-611
[89]余新天,黄海燕,付英斌,等.贝类毒素昆明种小鼠生物测定法建立[J].中国公共卫生,2009,25(5):590-591
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