• 陈修报,郑浩然,王洋,刘洪波,杨健.基于原代培养背角无齿蚌鳃细胞的镉毒性效应评价[J].环境科学学报,2020,40(7):2665-2670

  • 基于原代培养背角无齿蚌鳃细胞的镉毒性效应评价
  • Cytotoxicity assessment of cadmium on primary gill cell culture from Anodonta woodiana
  • 基金项目:中国水产科学研究院基本科研业务费(No.2019GH10);国家自然科学基金(No.31502166);江苏省自然科学基金(No.BK20161144);中国水产科学研究院淡水渔业研究中心基本科研业务费(No.2019JBFM05)
  • 作者
  • 单位
  • 陈修报
  • 中国水产科学研究院淡水渔业研究中心, 中国水产科学研究院长江中下游渔业生态环境评价和资源养护重点实验室, 无锡 214081
  • 郑浩然
  • 南京农业大学, 无锡渔业学院, 无锡 214081
  • 王洋
  • 南京农业大学, 无锡渔业学院, 无锡 214081
  • 刘洪波
  • 中国水产科学研究院淡水渔业研究中心, 中国水产科学研究院长江中下游渔业生态环境评价和资源养护重点实验室, 无锡 214081
  • 杨健
  • 1. 中国水产科学研究院淡水渔业研究中心, 中国水产科学研究院长江中下游渔业生态环境评价和资源养护重点实验室, 无锡 214081;2. 南京农业大学, 无锡渔业学院, 无锡 214081
  • 摘要:为了探究淡水贝类背角无齿蚌鳃细胞的原代培养途径,并阐释其在评价水体Cd2+毒性效应上的潜力,本研究比较了不同酶分解方法(链霉蛋白酶、胰蛋白酶)的鳃细胞存活率差异,并分析了L-15培养基中不同血清浓度(10% FBS、20% FBS)对鳃细胞存活率的影响,细胞置于20℃生化培养箱中培养.进而根据Cd2+对鳃细胞的LC25值设定0.0625、0.125、0.25、0.5和1.0 mg·L-1 5个Cd2+理论浓度梯度,对原代培养的鳃细胞进行24 h暴露,分析了细胞活力、总超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和酸性磷酸酶(AcP)的变化特征.结果表明:0.025%链霉蛋白酶在4℃分解16 h的鳃细胞存活率为98.2%±0.2%,显著高于0.25%胰蛋白酶在26℃分解30 min的89.4%±3.5%鳃细胞存活率(p<0.05);L15培养基加入10% FBS的细胞存活率总体显著高于添加20% FBS的细胞存活率(p<0.05).在上述较佳的分解和血清浓度组合条件下,细胞培养120 h后,其存活率仍高达90.1%±4.7%.鳃细胞活力随着Cd2+浓度的增加而降低,两者之间呈显著的线性负相关(p<0.05);随着Cd2+浓度的增加,SOD和AcP含量总体增加,而CAT含量呈现出"诱导-抑制"的变化趋势.本研究初步建立了较为适宜的背角无齿蚌鳃细胞的原代培养方法,并揭示了其原代培养鳃细胞的细胞活力及SOD、CAT、AcP水平,具有作为评价水环境Cd2+毒性/污染的生物指标的潜力.
  • Abstract:This study was aimed to effectively investigate an approach on development of primary gill cell culture for freshwater mussel Anodonta woodiana, and assess the potential of the cell on cytotoxicity of aquatic Cd2+ in vitro. The survival rates of the gill cells, cultured in an incubator at 20℃, were firstly evaluated by different dissociation methods with pronase and trypsin, or L-15 media culture with 10% FBS and 20% FBS serum concentrations. Then, the cells were used in vitro testing on the variations of cell viability, levels of total superoxide dismutase (SOD), catalase (CAT), acid phosphatase (AcP) at Cd2+concentration gradients (0, 0.0625, 0.125, 0.25, 0.5 and 1.0 mg·L-1) for 24 h exposure, based on the corresponding LC25 data. The cell survival rate 98.2%±0.2% dissociated by 0.025% pronase at 4℃ for 16 h was significantly higher than that 89.4%±3.5% dissociated by 0.25% trypsin at 26℃ for 30 min (p<0.05). The survival rate of the cultured cells in L-15 media with 10% FBS was obviously higher than that with 20% FBS (p<0.05). The best survival rate of the cell obtained in this study was as high as 90.1%±4.7% after 120 h culture at the abovementioned better dissociation and FBS conditions. Additionally, the cell viability decreased with the increase of Cd2+ concentration, presenting a significant linear negative correlation (p<0.05). In contrast, the levels of SOD and AcP were generally increased, while that of CAT showed an induced inhibition trend. The present study preliminarily developed a suitable primary culture approach for the gill cells of A. woodiana, and revealed the corresponding potential for in vitro Cd2+ cytotoxicity assessment using cell viability, and SOD, CAT, AcP levels as biomarkers.

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