纳米硫化镉对大鼠肾小管上皮细胞膜的影响

Effects of cadmium sulfide nanoparticles on membrane of rat renal tubular epithelial cells

目的研究纳米硫化镉(nCds)对大鼠肾小管上皮(NRK-52E)细胞膜的损伤作用。方法将NRK-52E细胞分别暴露于含终浓度为0(对照)、5、10、15、20、30μg/ml nCds的培养基中染毒24 h。检测细胞的增殖活性;采用双嵌入剂乙锭均二聚物(EthD-1)和钙黄绿素(Calcein AM)对细胞进行双染色后,荧光显微镜观察整体细胞膜损伤情况;采用双激光共聚焦显微镜荧光染色观察单个细胞膜损伤情况;透射电子显微镜定位细胞内的nCds。结果与对照组比较,随着nCds染毒浓度的升高,NRK-52E细胞的存活率呈下降趋势,10~30μg/ml nCds染毒组细胞的存活率均较低,差异有统计学意义(P<0.05)。EthD-1/Calcein AM双染色结果显示染毒剂量的升高导致细胞膜破损增加,10μg/ml nCds使细胞膜破损的细胞数明显增多。激光共聚焦结果显示随着nCds的剂量增加,细胞膜的荧光强度降低,细胞膜受损。透射电子显微镜观察到,对照组细胞结构完整,细胞膜平滑;nCds染毒组观察到nCds颗粒在细胞膜外聚集,细胞膜有破损情况出现,nCds颗粒进入到细胞质内。结论 nCds可通过破坏细胞膜的方式进入到细胞质中,从而抑制NRK-52E细胞的生长。

Objective To understand the damage of the membrane of rat renal tubular epithelial cell line(NRK-52E) exposed to nano-cadmium sulfide particles(nCds). Methods NRK-52E cells were respectively exposed to nCds in medium at the doses of 0(control),5,10,15,20,and 30 μg/ml respectively for 24 hours and the proliferation activity of the cells was detected. After double-staining with EthD-1 and Calcein AM,the whole cell membrane damage was observed by fluorescence microscopy. Single cell membrane damage was observed by dual-laser confocal microscopy fluorescence staining and nCds in cells were localized by transmission electron microscopy. Results Compared with the control group,the survival rate of NRK-52E cells showed a downward trend with the increase of nCds concentration,and the survival rate of cells exposed to 10-30 μg/ml nCds was significantly lower(P<0.05). EthD-1/Calcein AM double staining showed that the cell membrane damage increased with the increase of the exposure doses,and the number of cells damaged by 10 μg/ml nCds increased significantly.Laser confocal imaging showed that as the doses of nCds increased,the fluorescence intensity of the cell membrane decreased,which suggested that the cell membrane was damaged. Transmission electron microscopy imaging presented intact cellular structure and smooth cytomembrane in control group,as well as accumulation of nano-cadmium sulfide particles in cytoplasm and outside the cell membrane,breakage of cytomembrane in exposure groups. Conclusion Nano-cadmium sulfide particles can enter the cytoplasm through disrupting the cell membrane,thereby inhibit the growth of NRK-52E cells.