Primary Culture Technique Of Oyster (Crassostrea Hongkongensis) Gill Cells And Application Of The Cells In Molecular Toxicology

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.