不同粒径纳米硒化铅对大鼠胚胎神经干细胞的氧化损伤作用

Oxidative damages induced by lead selenide nanoparticles with different sizes in embryonic neural stem cells of rat

目的探讨不同粒径纳米硒化铅(Pb Se)对大鼠胚胎神经干细胞(NSCs)的氧化损伤作用。方法将处于对数生长期的NSCs暴露于终浓度分别为0(对照)、6.25、12.5、25、50、75、100、200μg/ml两种粒径(8、25 nm)的纳米Pb Se悬液24 h,采用MTT法检测细胞存活率,采用乳酸脱氢酶(LDH)法检测细胞膜损伤情况。将处于对数生长期的NSCs暴露于终浓度分别为0(对照)、60、80、100μg/ml两种粒径的纳米Pb Se悬液24 h,检测细胞内超氧化物歧化酶(SOD)、谷胱甘肽过氧化物还原酶(GSH-Px)活力及谷胱甘肽(GSH)、丙二醛(MDA)含量,采用ELISA法检测细胞培养液中8-羟基-2'-脱氧鸟嘌呤核苷(8-OH-d G)的含量。结果与对照组相比,不同浓度的两种粒径纳米Pb Se暴露组NSCs细胞的存活率均降低,差异有统计学意义(P<0.05);且随着两种粒径纳米Pb Se暴露浓度的升高,NSCs细胞的存活率均呈下降趋势。当纳米Pb Se暴露浓度≥50μg/ml时,8 nm粒径纳米Pb Se暴露组NSCs细胞的存活率均低于25 nm粒径纳米Pb Se暴露组,差异有统计学意义(P<0.05)。与对照组相比,不同浓度两种粒径纳米Pb Se暴露组NSCs细胞培养液中的LDH活力均升高,差异有统计学意义(P<0.05);且随着两种粒径纳米Pb Se暴露浓度的升高,NSCs细胞培养液中的LDH活力均呈上升趋势。在相同浓度下,8 nm粒径纳米Pb Se暴露组NSCs细胞培养液中LDH的活力均高于25 nm粒径纳米Pb Se暴露组,差异有统计学意义(P<0.05)。与对照组相比,不同浓度两种粒径纳米Pb Se暴露组NSCs细胞上清液中SOD、GSH-Px活力和GSH含量均降低,而MDA含量均升高,差异均有统计学意义(P<0.05);且随着两种粒径纳米Pb Se暴露浓度的升高,NSCs细胞上清液中SOD、GSH-Px活力和GSH含量均呈下降趋势,而MDA含量均呈上升趋势。与相同浓度25 nm粒径纳米Pb Se暴露组比较,各浓度8 nm粒径纳米Pb Se暴露组NSCs细胞上清液中SOD、GSH-Px活力和GSH含量均降低,而MDA含量均升高,差异均有统计学意义(P<0.05)。与对照组相比,不同浓度两种粒径纳米Pb Se暴露组NSCs细胞培养基中8-OHd G的含量均升高,差异有统计学意义(P<0.05);且随着两种粒径纳米Pb Se暴露浓度的升高,NSCs细胞培养基中8-OHd G的含量均呈上升趋势。与相同浓度25 nm粒径纳米Pb Se暴露组比较,80、100μg/ml的8 nm粒径纳米Pb Se暴露组NSCs细胞培养基中8-OH-d G含量均升高,差异有统计学意义(P<0.05)。结论纳米Pb Se引起NSCs细胞的氧化损伤可能是导致其产生细胞毒性的主要原因。

Objective To understand the oxidative damages induced by different sizes of lead selenide nanoparticles（nano Pb Se） in embryonic neural stem cells（NSCs） of rat. Methods NSCs cells were exposed to nano Pb Se with two sizes（8 and25 nm） at the doses of 6.25, 12.5, 25, 50, 75, 100 and 200 μg/ml respectively for 24 h. The cell viability was observed by MTT assay and the cell membrane integrity was detected by cell culture medium of LDH activity. After NSCs cells were exposed to nano Pb Se with two sizes at different concentrations（60, 80 and 100 μg/ml） respectively for 24 h,the activities of SOD,GSH-Px and the contents of GSH,MDA inside cells were measured and 8-OH-d G levels in the medium were determined using ELISA. Results Compared with the control group,the viabilities of NSCs in nano Pb Se exposure groups decreased with increasing concentration of nano Pb Se with two sizes（P〈0.05）. When concentration of nano Pb Se was more than 50 μg/ml,the viabilities of NSCs in 8 nm nano Pb Se exposure group were lower than that in the same concentration of 25 nm nano Pb Se exposure group. Compared with the control group, LDH activities in NSCs cell culture medium in nano Pb Se exposure groups increased with increasing concentration of nano Pb Se with two sizes（P〈0.05）. At the same concentration, LDH activities in NSCs cell culture medium in 8 nm nano Pb Se exposure group were higher than that in 25 nm nano Pb Se exposure group（P〈0.05）. After exposure to the two sizes of nano Pb Se with two sizes for 24 h, activities of SOD, GSH-Px and GSH contents of exposure groups were lower than those of control group, and MDA contents were higher than those of control group（P〈0.05）.As the concentration increased, the SOD, GSH-Px activities and GSH contents decreased in all exposure groups, whihe MDA contents increased（P 0.05）. The activities of SOD, GSH-Px and GSH contents of each concentration of 8 nm nano Pb Se exposure group were lower than those of the same concentration of 25 nm nano Pb Se exposure groups, and MDA contents of 8 nm nano Pb Se exposure group were higher than that of the same concentration of 25 nm nano Pb Se exposure group（P〈0.05）.Compared with the control group, the contents of 8-OH-d G in NSCs cell culture medium in each exposure group increased with increasing concentration of nano Pb Se（P〈0.05）. 8-OH-d G levels in the medium of 80 and 100 μg/ml 8 nm nano Pb Se exposure groups were higher than those of the same concentration of 25 nm nano Pb Se exposure groups（P〈0.05）. Conclusion The oxidative damage may be the primary cause for the cytotoxicity of nano Pb Se.