Vero细胞对2,4,6-三氯苯酚的毒性响应特征和敏感性分析

以2,4,6-三氯苯酚(2,4,6-trichlorophenol,TCP)为例,采用MTT比色实验和ANNEXIN V-FITC/PI双染色-流式细胞技术研究了Vero细胞对有机化学污染物的毒性响应特征和敏感性,以期发现一种灵敏、可靠的表征微量化学物质综合毒性的方法.研究结果表明:1)TCP浓度对Vero细胞的毒性响应特征有决定作用.低浓度TCP(≤0.5mg·L-1)即可使部分细胞从原有的刚性不规则三角形结构变为圆形或椭圆形,但细胞的生长未受明显抑制.TCP浓度在1~5mg·L-1时,细胞生长开始受到抑制,表现为细胞增殖受到抑制及部分细胞凋亡或死亡,但增殖抑制率和TCP浓度之间的关系不明显.当TCP浓度大于5mg·L-1时,细胞增殖明显受到抑制,且随TCP浓度的增加,细胞增殖抑制率逐渐升高.2)TCP作用时间对Vero细胞的毒性响应特征有显著影响.ANNEXIN V-FITC/PI双染色-流式细胞仪检测结果表明,TCP作用24h内Vero细胞毒性表现为以细胞膜的完整性受到损伤为主,凋亡或坏死细胞比例较小;而48h后,凋亡细胞的比例明显增加.细胞复壮实验结果进一步证实24h内形态发生变化的细胞并未完全坏死或凋亡.以上结果表明细胞形态变化和细胞膜损伤均为细胞毒性的早期表现特征,两者之间可能存在某种相关性,以此表征化学污染物的生物毒性具有较高的灵敏度,值得进一步研究.

Cytotoxic Response Characteristics and Sensitivity in Mammalian Vero Cells Exposed to 2,4,6-Trichlorophenol

In order to find a sensitive and reliable method to evaluate comprehensive toxicity of trace level chemicals in environment, MTT assay and ANNEXIN V-FITC/PI-Flow Cytometry are used to study the cytotoxic response characteristics and sensitivity in Vero cells exposed to 2, 4, 6-trichlorophenol (TCP), a typical organic pollutant. Results show that: 1) TCP concentration plays a key role in determining the response characteristics of Vero cytotoxicity. Low dose of TCP(≤0.5mg·L-1) can cause cytomorphosis(some Vero cells become oval or circular from irregular triangle), while the growth of cells is not inhibited significantly. When TCP concentrations are 1~5mg·L-1, the growth of cells is inhibited, which mainly manifested as inhibition of proliferation and apoptosis or death of some cells. The relationship between the inhibition of proliferation and the TCP concentrations is not obvious. When TCP concentrations are more than 5mg·L-1, significant inhibition of proliferation is found. With the increase of TCP concentrations, the rate of inhibition of proliferation increase gradually. 2) Exposure time has obvious effects on response characteristics of Vero cytotoxicity. Results of ANNEXIN V-FITC/PI-Flow Cytometry show that: cytotoxicity of Vero cells is mainly manifested as the damage of cell membrane intact, and the proportions of cell apoptosis and necrosis are low in 24h exposure of TCP. After 48h exposure of TCP, the proportion of cell apoptosis increases significantly. The results of cell rejuvenation further confirm that the cells which morphology changes within 24h are not complete necrosis and apoptosis. The above results suggest that both of the change of cell morphology and the membrane damage should be the early response characteristics of Vero cytotoxicity and there is certain pertinence between them, which show high sensitivity in characterizing the biological toxicity of chemicals.