栉孔扇贝鳃细胞原代培养与B[α]P细胞毒性检测技术的研究

Study on the primary culture of gill cells and B[α]P cytotoxicity detection technology in the scallop Chlamys farreri

中文关键词: 栉孔扇贝  鳃细胞  原代培养  B[α]P  细胞毒性

英文关键词: Chlamys farreri  Gill cells  Primary culture  B[α]P  Cytotoxicity

中文摘要:

      本研究优化了栉孔扇贝(Chlamys farreri)鳃细胞制备方法和原代培养条件，采用3种细胞活性检测技术，比较分析了苯并(a)芘(B[α]P)对栉孔扇贝鳃细胞的毒性作用。结果显示，栉孔扇贝鳃组织在1%青霉素–链霉素(双抗)和庆大霉素消毒10~30 min内，原代培养鳃细胞存活率无明显差异。消毒30 min时，镜检无染菌现象，细胞状态良好。胰蛋白酶消化时间对鳃细胞收获量影响显著，在消化15~25 min内，鳃细胞存活率较高，胰蛋白酶的最佳消化时间为25 min。在150~300 g相对离心力作用下，鳃细胞形态和存活率存在明显差异，在150 g时，存活率和细胞完整性较好。添加胎牛血清(5%~20%)在6~12 h内鳃细胞存活率无显著变化，培养24 h时，5%和20%处理组存活率显著下降，10%和15%处理组存活率无明显变化。台盼蓝拒染法细胞活性检测表明，B[α]P对栉孔扇贝鳃细胞活性无明显影响；CCK8试剂法得出，仅在16 μg/mL最高浓度下，鳃细胞活性受到显著抑制；而中性红比色法显示，鳃细胞毒性作用与B[α]P浓度、染毒时间呈正相关性。研究表明，栉孔扇贝鳃细胞最佳制备方法：消毒30 min、胰蛋白酶消化25 min、相对离心力为150 g、原代培养基添加10%胎牛血清为最佳。同时，中性红比色法可以作为评价B[α]P对栉孔扇贝鳃细胞毒性的敏感指标。

英文摘要:

      In this study, the preparation method and primary culture conditions of Chlamys farreri gill cells were optimized. The toxic effects of benzo[a]pyrene (B[α]P) were compared and analyzed by three cell activity detection techniques. The results showed no significant difference in the survival rates of the primary cultured gill cells disinfected with 1% penicillin-streptomycin solution (Pen-Strep) and gentamicin for 10~30 min. There were also no bacterial infections observed in the microscopic examinations after 30 min, and the cells were in good condition. The effect of trypsin digestion time on the harvest of gill cells was significant. Within 15~25 min of digestion, the survival rate of the gill cells was higher, and the best trypsin digestion time was 25 min. With 150~300 g relative centrifugal force, the morphology and survival rate of the gill cells significantly differed, and the survival rate and cell integrity were better at 150 g. There were no changes in survival within 6~12 h after adding fetal bovine serum (FBS, 5%~20%). At 24 h, the survival rates of the 5% and 20% treatment groups decreased significantly, but the 10% and 15% treatment groups were unaffected. The cytotoxicity of B[α]P on the scallop gill cells was detected by three cell activity tests, and the results showed no change in the activity of the gill cells with the trypan blue exclusion assay. The cell counting kit-8 assay showed that the activity of the gill cells was significantly inhibited at the highest concentration (16 μg/mL), while the neutral red assay showed a positive toxicity correlation between the B[α]P concentration and time. These results suggest that the best preparation method for C. farreri gill cells is disinfection for 30 min, trypsin digestion for 25 min, relative centrifugal force at 150 g, and the addition of 10% fetal bovine serum to the primary culture medium. The neutral red assay can be used as a sensitive index to evaluate the toxicity of B[α]P.