可溶性铀的内吞外排及对HK-2细胞的损伤研究

Cellular endocytosis and exocytosis of soluble uranium and its damage to HK-2 cells

目的研究人肾近曲小管细胞(HK-2)对可溶性铀的内吞和外排情况,了解铀暴露后的细胞毒性效应。方法采用CCK-8法测定不同浓度铀染毒后细胞活性,分别在光镜和透射电子显微镜下观察细胞铀染毒后的变化;采用ICP-MS法测定细胞在不同时间对铀的内吞、外排情况;采用流式细胞仪检测铀染毒后细胞周期和细胞凋亡的变化。结果 HK-2细胞在铀暴露后出现剂量依赖性损伤,细胞周期阻滞在G1期,细胞发生凋亡和坏死,细胞增殖受到了抑制;细胞内吞的铀含量在24 h内逐渐增加,同时铀的内吞量存在阈值,细胞表面结合的铀含量分数较低(<0.2%),细胞内吞的铀1 h内40%以上都会排出胞外;铀在细胞内和细胞外均会形成针样状沉淀(300μM);结论细胞在铀染毒后出现周期阻滞、凋亡和细胞活性下降,可溶性铀的内吞、外排过程非常迅速,细胞可将可溶性铀转化为无毒性的沉淀。

Objective To investigate the endocytosis and exocytosis of soluble uranium in human kidney proximal tubular epithelial(HK-2)cells and the cytotoxicity after uranium exposure.Methods Cell Counting Kit-8 assay was used to determine the cell viability after different concentrations of uranium exposure,and optical microscopy and transmission electron microscopy were used to observe the changes in cells after uranium exposure.Inductively coupled plasma mass spectrometry was used to monitor the endocytosis and exocytosis of uranium over time by cells.Flow cytometry was used to assess the changes in cell cycle and apoptosis after uranium exposure.Results After uranium exposure,HK-2 cells showed dosedependent damage;cell cycle was arrested in G1 phase;cell apoptosis and necrosis occurred;cell proliferation was inhibited.The content of endocytic uranium increased gradually within 24 h,and there was a threshold for uranium endocytosis,while the fraction of uranium binding to cell surface was low(<0.2%).Over 40%of the endocytic uranium would be exocytosed within 1 h.Uranium could form needle-like precipitates in both intracellular and extracellular areas after uranium exposure.Conclusion After uranium exposure,cells show decreased viability,cell cycle arrest,and cell apoptosis.The process of endocytosis and exocytosis of soluble uranium is very rapid.HK-2 cells can convert soluble uranium into non-toxic precipitates.