【作者】 黄爱君；

【导师】 杨汝德； 刘建军；

【作者基本信息】 华南理工大学 ， 生物化工， 2010， 硕士

【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。更多还原

【Abstract】 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.更多还原