栉孔扇贝血细胞活性检测方法的筛选与应用

Screening and Application of the Haemocyte Viability Detection Methods of Scallop Chlamys farreri

筛选栉孔扇贝（Chalmys farreri）血细胞活性的检测方法,研究苯并[a]芘（B[α]P）对栉孔扇贝血细胞活性的影响。结果表明96孔培养板中栉孔扇贝血细胞接种密度在（1-32）×10^4cell/孔时,血细胞活性无显著性差异（P〉0.05）,且细胞存活率在97.42%-98.82%之间,台盼蓝排斥法、四甲基偶氮唑（MTT）、二甲氧唑黄（XTT）比色法、乳酸脱氢酶（LDH）活性测定法均可作为栉孔扇贝血细胞活性的测定方法,而当血细胞接种密度在（12-32）×10^4/孔时,中性红比色法亦可作为血细胞活性的检测方法;同时实验采用Eagle培养液进行血细胞原代培养在0-24 h内血细胞活性较好。采用中性红比色法检测B[α]P对栉孔扇贝血细胞活性具有显著抑制作用,且抑制程度与B[α]P作用浓度、时间呈正相关,其余方法均未检测出B[α]P对血细胞活性的影响。由此可见,B[α]P对栉孔扇贝血细胞活性具有免疫毒性作用,中性红比色法可以作为评价B[α]P对栉孔扇贝血细胞免疫活性的敏感指标。

In this work, the suitable detection methods of haemocyte viability and the effects of B[α]P on the haemocyte viability of scallop Chlarnys farreri were studied. The results showed that when the haemocyte density in a 96-well microplate was between（1-32） × 10^4 cells per well, the livability of haemocytes was high（as high as 97.42%- 98.82% ）, and the MTT-, XTT-and LDH-assays were all suitable viability detection methods of haemocytes. While only the haemocyte density was between（12-32）× 10^4 cells per well, the neutral red assay was a suitable detection method. The cell viability of the primary culture haemocytes in 24 h was good using Eagle culture medium. In B[α]P exposure experiment, except that the neutral red assay showed that B [ α]P significantly inhibited the haemocyte viability, the other methods did not detect the inhibition effect, besides, the results of the neutral red assay showed that the inhibition degree had a positive correlation with the concentration and exposure time of B[α]P. So it is concluded that the B[α]P had immunotoxicity effects on haemocytes and the neutral red assay could be used as a sensitive index to evaluate the effect of B[ α] P on the haemocyte viability of scallop C. farreri. K