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氧化应激体外模型的构建及沙利度胺保护作用的研究

Construction of oxidative stress model in vitro and observation on the protective effect of thalidomide on oxidative stress
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摘要 目的:通过构建体外氧化应激损伤模型,探讨沙利度胺(Thalidomide)的保护作用以及最佳的作用时间点和浓度。方法:体外氧化应激损伤模型采用脂多糖(LPS)构建,流式细胞术测定LPS最佳造模时间及浓度。MTT法测定沙利度胺的无细胞毒性浓度。流式细胞术测定沙利度胺最佳预处理时间及不同浓度沙利度胺的保护作用。结果:MTT结果显示沙利度胺浓度在0~125 mg/L范围内对人脐静脉内皮细胞(HUVEC)基本是无损伤的,故选取沙利度胺最大浓度作为后续试验的浓度。流式细胞术结果显示,较低浓度的LPS即可引起凋亡,与处理6、12 h相比,0.25、0.5 mg/L LPS分别处理HUVEC 24 h后的凋亡率增加(P<0.05),且1 mg/L LPS处理HUVEC 24 h后凋亡率最高达到92%,相比处理6 h和12 h后的凋亡率,差异有统计学意义(P<0.05)。2 mg/L LPS处理HUVEC在各个时间点与1 mg/L LPS处理后的凋亡率基本没有差别。100 mg/L沙利度胺预处理6 h后凋亡率比模型组低,差异有统计学意义(P<0.05),但是与预处理12、24 h相比,凋亡率基本没有改变,差异无统计学意义(P>0.05)。此外,100 mg/L沙利度胺预处理后凋亡率(31.5%)相比10 mg/L(52.6%)和1 mg/L(68.6%)的沙利度胺预处理,凋亡率下降,差异有统计学意义(P<0.05)。结论:氧化应激损伤模型已成功构建,可用于进一步研究;沙利度胺可以保护LPS诱导的氧化应激下的细胞损伤,为沙利度胺进一步应用于临床提供了很好的依据。 Objective:To observe the protective effect of thalidomide as well as its optimal time and concentration on the oxidative stress injury model developed in vitro.Methods:Oxidative stress injury model was constructed in vitro technique with lipopolysaccharides(LPS),and flow cytometry was used to determine the optimal time and concentration of LPS.MTT assay was performed to determine the cytotoxic concentration of thalidomide.Then flow cytomertry was used to determine the optimal pretreatment time of thalidomide and the protective effect of different concentration of thalidomide on the injury model.Results:MTT assay showed that thalidomide concentration in between 0 and 125 mg/L produced no cytotoxic effect on human umbilical vein endothelial cells(HUVECs),for which the maximum concentration of thalidomide was selected as the reference for subsequent tests.Flow cytometry showed that low concentration of LPS had led to apoptosis of HUVECs,and apoptosis rate was significantly increased for HUVECs treated by LPS in dose of 0.25 mg/L and 0.5 mg/L after 24 h compared to the treatment after 6 h and 12 h(P<0.05),and the apoptosis rate of HUVECs was up to 92%at 24 h after LPS treatment in dose of 1 mg/L.The difference was significant in apoptosis rate as compared to the rate at 6 and 12 h after LPS treatment(P<0.05),yet was insignificant compared to the rate at 12 and 24 h following treatment(P>0.05).The apoptosis rate of HUVECs had no difference between LPS treatment in dose of 2 mg/L and 1 mg/L at different time point.The apoptosis rate was significantly lower than that of model group at 6 h after pretreatment with thalidomide in dose of 100 mg/L(P<0.05),yet remained no obvious change as compared to the rate at 12 and 24 h(P>0.05).In addition,the apoptosis rate of HUVECs was different by pretreatment with thalidomide in dose of 100 mg/L(31.5%)from that in dose of 10 mg/L(52.6%)and 1 mg/L(68.6%)(P<0.05).Conclusion:Oxidative stress injury model was successfully constructed,and can be used for following study purpose.Additionally,thalidomide can produce protective effect on cell injury from oxidative stress induced by LPS,which may be good basis for following research of thalidomide in clinical use.
作者 高红亮 吴超 王超 程倩 李曙 Gao Hongliang;Wu Chao;Wang Chao;Chen Qian;Li Shu(Department of Pathophysiology,Wannan Medical College,Wuhu 241002,China)
出处 《皖南医学院学报》 CAS 2018年第2期109-112,119,共5页 Journal of Wannan Medical College
基金 安徽省高校优秀青年人才支持计划重点项目(gxyq ZD2016172) 大学生创新训练计划(201510368038 201610368116)
关键词 沙利度胺 脂多糖 氧化应激 保护作用 thalidomide LPS oxidative stress protective effect
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