参环毛蚓肠上皮细胞重金属镉离子的耐受性研究

Study on Heavy Metal Cadmium Ion Tolerance of Pheretima aspergillum Intestinal Epithelial Cells

【目的】观察参环毛蚓肠上皮细胞(IEC)对重金属镉离子(Cd2+)的耐受能力,探讨其耐受特性与广地龙药材重金属超标的相关性。【方法】采用不同浓度Cd2+对参环毛蚓IEC进行胁迫处理,分别利用台盼蓝染色法和噻唑蓝法测定细胞死亡率和细胞活力,并借助显微镜观察与比较参环毛蚓IEC胁迫处理前后的形态变化。【结果】IEC对Cd2+的耐受特性主要表现为:①未经孵育时IEC可耐受Cd2+的最高浓度为8μmol/mL;②胁迫处理24 h后IEC可耐受Cd2+的最高浓度为6.25×10-2μmol/mL;③胁迫处理48 h时,IEC可耐受Cd2+的最高浓度为3.125×10-2μmol/mL,上述3个浓度分别是《中华人民共和国药典》(2010年版)中地龙项下镉限量指标的2 997.33、23.33和11.66倍;④IEC对Cd2+的耐受性在一定的时间范围内与剂量呈负相关。【结论】参环毛蚓IEC对重金属Cd2+具有较高的耐受性。Cd2+对IEC的毒性作用不是接触性的损伤,而有可能是产生于细胞代谢过程中。

Objective To analyze the tolerance of Pheretima aspergillum （E. Perrier） intestinal epithelial cells （IEC） on the heavy metal of cadmium ion （Cd2＋）, and to explore the correlation of Cd2＋ tolerance with heavy metal over the limit in herbal medicinal material of Pheretima aspergillum. Methods Pheretima aspergillum IEC were stressed by Cd2＋/at various concentrations. The death rate of IEC was observed after Trypan Blue staining, and the cell viability was determined by methyl thiazolyl tetrazolium （MTI＇） method. An optical microscope was applied for the observation of IEC morphological changes. Results The characteristics of IEC tolerance to Cd2＋ were mainly shown as follows： （1） The highest tolerated concentration of Cd2＋ for IEC was 81xmol/mL before IEC was stessed by Cd2＋. （2） After stressed with Cd2＋ for 24 h, IEC had the tolerance to the maximum tolerated concentration of Cd2＋ at 6.25 x 10-2 μmol/mL. （3） After stressed with Cd2＋ for 48 h, IEC had the tolerance to the maximum tolerated concentration of Cd2＋ at 3.125 x 10-2 μmol/mL. The above three concentrations were 2 997.33, 23.33, 11.66 times as much as the cadmium limit of Pheretima issued by 2010 edition of Chinese Pharmacopoeia. （4） The tolerance of IEC to Cd2＋ was negatively correlated with the dose within certain period. Conclusion The Pheretima aspergillum （E. Perrier） intestinal epithelial cells show certain tolerance to the high concentrations of cadmium ion. The toxicity of Cd2＋ to IEC is not contact injury, but probably occurs in the process of cell metabolism.