【作者】 王彬；

【导师】 张其中；

【作者基本信息】 暨南大学 ， 水生生物学， 2010， 硕士

【摘要】 近江牡蛎是我国南方的重要经济贝类,养殖产量高,主要分布在近岸海域和河口地区,这些地方是污染日益严重的区域,所以,近江牡蛎又是重要的污染指示生物。无论研究其作为经济贝类养殖过程中的病害控制和遗传育种等问题的基础分子机制,还是研究其作为污染指示种对污染物反应的分子毒理机制都需要近江牡蛎的培养细胞。本研究以近江牡蛎鳃为材料,摸索了几种培养鳃细胞的方法,并用所培养的鳃细胞进行了初步的分子毒理学实验。具体研究结果如下：采用改良的DMEM(HG)培养基,建立了近江牡蛎鳃细胞体外培养技术,包括鳃组织块培养法、胰酶消化培养法和胶原酶消化培养法。原代培养实验中,组织块培养法接种6h后,细胞开始从组织块中迁出,细胞形态较小,呈圆形、椭圆形或多边形,直径3～6μm。培养至3d时,细胞在组织块周围形成生长晕。培养至6d时可进行细胞传代,本次实验细胞已传至第6代。胰酶消化法接种约2h后,小部分细胞逐渐贴壁,从形态上主要分为两类,一类为小型细胞,形态为圆形、椭圆形或多边形,直径3～6μm,数量多,增殖速度较快；另一类为大型细胞,形态为圆形、椭圆形或多边形,直径10～20μm,部分细胞内部含有颗粒,数量较少,增殖速度较慢。培养至2d时可进行细胞传代,传代培养物中的优势细胞皆为小型细胞。本次实验细胞已传至第6代。胶原酶消化法接种约1h后,大部分细胞逐渐贴壁,形态与胰酶消化法获得的细胞类似,分为大小两种细胞。培养至2d时可进行细胞传代,传代培养物中的优势细胞皆为小型细胞。本次实验细胞已传至第6代。用两种不同浓度的CuSO4(10、100μg/L)分别处理培养的近江牡蛎鳃细胞,处理1h,恢复6h后,经RT-PCR扩增发现,能从10μg／L处理的近江牡蛎鳃细胞中扩增出清楚的257bp的HSP90条带,而对照的扩增条带很淡,从100μg／L处理的鳃细胞中也仅能扩增到模糊的条带。这说明10μg/L CuSO4能诱导HSP90表达,100μg/L CuSO4能损伤培养细胞,引起mRNA降解,扩增效果差。最终表明所培养的鳃细胞可用于分子毒理学研究。更多还原

【Abstract】 Crassostrea hongkongensis is one of important bivalves in South China Sea, and reared on a large scale, during culture period of this species, it was frequently disturbed by various diseases. Meanwhile, it distributes in the area near the coast and estuary area, which are polluted by various pollutants. Therefore, it has been used as environmental pollutant indicator species to monitor marine environmental quality. In order to further study both the molecular mechanism of the oyster against diseases and find an early warning molecular biomarker for monitoring early polluted status, it is necessary to get its culture cells at first. This study established several methods to culture the oyster gill cells, and made an attempt of molecular toxicology experiments with the cultured gill cells. The results are as follows:The gill cells derived from the oyster(Crassostrea hongkongensis) were cultured in vitro with the modified medium DMEM(HG) by means of three methods(gills tissue culture, trypsinization methods, and collagenase digestion method).In the gills tissue culture method, the small cells, which were rounded, elliptic or polygonal with diameter of 3～6μm, began to migrate out from the gills tissue at 6h post-inoculation. And the cell layers were present around the tissues at 3d post-inoculation, and could be subcultured at 6 days. In present study, the gill cells have been subcultured to the sixth generation. In trypsinization method, a small population of gill cells were adherent at 2 hours post-inoculation of trypsinization. The cells could be morphologically divided into two groups including small cells with diameter of 3～6μm and large cells with diameter of 10～20μm. The two group cells were all rounded, elliptic or polygonal, some of the large cells were granular cells. The small cells were dominant and more than the large cells in number. Being cultured for 2 days, the gill cells could be subcultured. In present study, the gill cells have been subcultured to the sixth generation. In collagenase digestion method, Most of gill cells were adherent at 1 hour post-inoculation of collagenase digestion. The cells are similar to the cells obtained by means of trypsinization including small cells and large cells. The small cells were dominant and more than the large cells in number. Being cultured for 2 days, the gill cells could be subcultured. In present study, the gill cells have been subcultured to the sixth generation.Crassostrea hongkongensis gill cells were treated with two concentrations of CUSO4 (10、100μg/L) for 1h and subsequently recovered for 6h. The results of RT-PCR amplification showed that 257bp band of HSP90 gene was amplified in most density from the cells treated with 10μg/L CUSO4, and in little density both with 100μg/L CuSO4 and in control. This indicate that 10μg/L CuSO4 could induce HSP90 expression, 100μg/L CUSO4 could damage the treated cells and caused the degradation of mRNA. The result suggested that the cultured gill cells can be used in molecular toxicology researches.更多还原