PM2.5中不同组分对人脐静脉内皮细胞的联合毒性作用研究

目的：探讨PM_2.5中各种组分单独以及联合暴露对于人脐静脉内皮细胞（HUVECs）的毒性作用，并探讨不同组分之间是否存在交互作用。方法：以永生化的HUVECs细胞为研究对象，分别给予0、5、10、20、40、80、160 μg/mL纳米炭黑颗粒（下称炭黑），0、2.5、5、10、20、40、80、160 μg/mL尘土颗粒（下称尘土），0、6.25、12.5、25、50、100、200、400 μmol/L醋酸铅，0、5、10、20、40、80、160 μmol/L亚砷酸钠和氯化镉，染毒24 h，采用CCK-8法测定不同受试物对细胞存活率的影响，并计算不同受试物对HUVECs细胞的半数抑制浓度（IC₅₀）。然后进行联合染毒实验，选用细胞存活率为80%时的剂量，炭黑（或尘土）分别与铅、砷、镉对HUVECs进行联合染毒24 h，用CCK-8测定细胞存活率；另外根据PM_2.5中各组分实际占比，按m_铅：m_{炭黑/尘土}=1：50，m_砷：m_{炭黑/尘土}=1：100，m_镉：m_{炭黑/尘土}=1：500的比例进行两两联合染毒，以及按m_铅：m_砷：m_镉：m_{炭黑/尘土}=10：5：1：500的比例进行四者联合染毒。染毒24 h后，用CCK-8法测定细胞存活率。结果：HUVEC细胞存活率随着炭黑、尘土、醋酸铅、亚砷酸钠、氯化镉染毒浓度的增加而下降（P<0.05），以上各受试物对HUVECs细胞的IC₅₀分别为16 μg/mL、110 μg/mL、184 μmol/L、15 μmol/L、14 μmol/L。炭黑与醋酸铅、亚砷酸钠、氯化镉联合染毒时，细胞存活率比炭黑单独染毒时分别下降了17.8%、43.8%、41.2%；尘土与醋酸铅、亚砷酸钠、氯化镉联合染毒时，细胞存活率比尘土单独染毒时分别下降了11.6%、27.8%、28.3%。联合染毒时细胞存活率比单独染毒时明显下降（P<0.05）。炭黑与亚砷酸钠、炭黑与氯化镉之间存在交互作用（均为P<0.05）。结论：PM_2.5中不同组分均可以对血管内皮细胞产生毒性作用，单独暴露时炭黑的细胞毒性作用比尘土大；但与3种金属联合暴露时，尘土比炭黑的联合暴露细胞毒性更大。铅、砷、镉对细胞的毒性作用大小排序为氯化镉>亚砷酸钠>醋酸铅，且炭黑颗粒与亚砷酸钠、炭黑颗粒与氯化镉之间存在交互作用，表现为协同作用。

Combined toxicity of different PM2.5 components in human umbilical vein endothelial cells

OBJECTIVE: To investigate toxic effects of various components in PM_2.5 and of their combined exposures on human umbilical vein endothelial cells (HUVECs),and to determine their interactive effects. METHODS: Immortalized HUVECs were exposed in culture to 0,5,10,20,40,80,160 μg/mL carbon black,0,2.5,5,10,20,40,80,160 μg/mL dust,0,6.25,12.5,25,50,100,200,400 μmol/L lead acetate,0,5,10,20,40,80,160 μmol/L sodium arsenite and cadmium chloride for 24 h. The CCK-8 assay was used to determine cell viability and IC₅₀ values were calculated. For the combined toxicity study,the doses at which cell viability was 80%,carbon black (or dust) was combined with lead,arsenic,and cadmium for 24 h,and the CCK-8 assay was used to determine cell viability. Additionally,according to the actual proportion of each fraction in PM_2.5,carbon black (or dust) was proportionally (m_lead:m_arsenic:m_cadmium:m_{carbon black/dust}=10:5:1:500) combined with lead,arsenic,and cadmium,respectively,for 24 h,and the CCK-8 assay was used to determine cell viability. RESULTS: The cell viability test shows a decreasing trend with increasing doses of carbon black,dust,lead acetate,sodium arsenite,and cadmium chloride (P<0.05),with IC₅₀ of 16 μg/mL,110 μg/mL,184 μmol/L,15 μmol/L,and 14 μmol/L,respectively. Cell viability was decreased by 17.8%,43.8%,41.2% when carbon black was combined with lead acetate,sodium arsenite,and cadmium chloride,respectively,and by 11.6%,27.8%,28.3% when dust was combined with lead acetate,sodium arsenite,and cadmium chloride,respectively. When carbon black and dust were combined with lead acetate,sodium arsenite,and cadmium chloride,the cell viability was significantly lower than when they were exposed individually (P<0.05). There were interactions between carbon black and sodium arsenite or cadmium chloride (all P<0.05). CONCLUSION: Different components in PM_2.5 can cause toxic effects to the vascular endothelial cells in vitro,the cytotoxic effect of carbon black was greater than dust particles. Upon combined exposure,dust was more cytotoxic upon simultaneous exposure to the other three metals. The order of toxic effect of lead,arsenic and cadmium was cadmium chloride > sodium arsenite > lead acetate,and the interactions between carbon black particles and sodium arsenite and cadmium chloride wa synergistic.