原代肝细胞和传代细胞对微囊藻毒素-LR的敏感性  

The cytotoxicity of microcystin-LR on primary rat hepatocytes and continuous cell lines

作　　者：谷康定[1] 林群馨[2] 

机构地区：[1]华中科技大学同济医学院公共卫生学院环境医学研究所,湖北武汉430030 [2]香港城市大学生物学与化学系

出　　处：《毒理学杂志》2005年第3期172-174,共3页Journal of Toxicology

基　　金：国家自然科学基金(20277013)

摘　　要：目的比较微囊藻毒素-LR对原代肝细胞和多种传代细胞株的毒性,探讨建立经济、简便的研究该毒素细胞模型的可能性。方法采用胶原蛋白酶2步灌流法制备大鼠原代肝细胞,以及用不同宿主来源的8种细胞株与不同浓度的微囊藻毒素-LR作用,MTT法测定细胞毒性终点及乳酸脱氢酶试验测定细胞膜受损与否。结果原代肝细胞培养至24h时,毒素浓度在5.0ng/mL即对细胞开始产生毒性并呈剂量-反应关系。48h时,细胞毒性更加剧烈,反应曲线呈典型S形,EC50为5.2ng/mL。当藻毒素为1.3ng/mL时,24h后,原代肝细胞LDH逸出开始随剂量增加而不断上升,20.0ng/mL乳酸脱氢酶逸出达到90%以上,细胞膜严重受损。受试8种传代细胞株无论从剂量、反应时间上都不如原代肝细胞敏感,其中相对敏感的KB细胞在培养96h,毒素浓度大于18.8μg/mL时,开始呈现明显剂量-反应关系,与原代肝细胞相差3个数量级。结论原代肝细胞是研究微囊藻毒素-LR急性毒性的理想模型,而KB细胞在较高剂量下可能是研究该毒素的传代细胞模型,其他传代细胞不适宜作为该毒素研究的模型。Objective The toxicities of microcystin-LR on primary rat hepatocytes and 8 continuous cell lines were investigated to compare their sensitivity to the toxin and to select the appropriate cell lines as models for further study. Method The isolated primary rat hepatocytes and the continuous cell lines were mixed with various concentrations of purified microcystin-LR in 96 well microtitre plates and incubated for 0, 6, 12, 24, 48, 72, and 96 h; meanwhile morphological alterations, colorimetric 3-（4,5-dimethylthiazol-2-yl）-2, 5-dephenyhetrazolium bromide （MTT） assays, lactate dehydrogenase （LDH） release assay were respectively used to evaluate the end point of toxicity. Results Microcystin-LR at level of 5.0 ng/mL exhibited toxic effects on the primary rat hepatocytes after 24 h incubation. Typical S-pattern of dose-response curve for the 48 h incubation hepatocyte with EC50 of 5.2 ng/mL was observed and the leakage of LDH from primary rat hepatocytes reached the highest level above 90% at 20 ng/mL of the toxin. Among 8 continuous cell lines, only KB cell line showed reliable significant dose-response effect at toxin concentrations greater than 18.8 /μg/mL with 96 h incubation. In KB cells the toxin induced marked morphological alterations at a concentration of about 37.5 μg/mL after incubation for 8 hours and significant amounts of LDH were released from the cells incubated for 72 h at concentrations of 18.8μg/mL and higher. Conclusion Primary rat hepatocyte is a sensitive cell model for detection of acute toxicity of microcystin-LR and the continuous KB cell line might be suitable for long-term and a little high dose exposure study on the toxin. Other subjected cell lines are not good materials for evaluating cytotoxicity of microcystin-LR.

关 键 词：原代肝细胞 传代细胞 微囊藻毒素-LR 细胞毒性 

分 类 号：R994.6[医药卫生—毒理学]