介质阻挡放电等离子体诱导白血病细胞凋亡及其机制研究

摘要

目的 探索介质阻挡放电(DBD)等离子体对肿瘤细胞的杀伤作用并分析其凋亡机制.方法 利用噻唑蓝(MTT)检测低温等离子体在不同作用时间下对正常脾脏白细胞和大鼠急性早幼粒白血病细胞(LT-12)细胞毒性的影响,检测等离子体处理后细胞内活性氧(ROS)含量的变化,利用Annexin V/PI双染法检测不同作用时间剂量下细胞凋亡情况,实时荧光定量PCR和Western Blot检测相关凋亡基因和蛋白的表达变化.结果 MTT结果表明,等离子体对细胞的杀伤呈作用时间剂量和时间依赖性,随着作用时间从30 s到240 s,处理8h后细胞的存活率从98％降至63％.在相同作用时间下,如240 s,细胞的存活率从2h的78％降至24 h的39％;Annexin V/PI双染法结果显示,等离子体作用可引起细胞凋亡,凋亡率不仅与等离子体作用时间剂量呈正相关,还与作用后的时间相关,等离子体处理后时间越长,其凋亡率越高,如处理12h后,细胞的凋亡率从60 s的48％增至120 s的55.3％;流式细胞仪检测的ROS的产生也显示了时间相关性,等离子体作用后其ROS立即增至1.24倍,至20 h急剧增高至5.39倍;qRT-PCR和Western Blot的实验结果表明,在等离子体处理后8～12 h Caspase家族和Bcl-2家族基因和蛋白表达非常活跃.结论 本研究表明低温等离子体能有效杀伤肿瘤细胞,凋亡是其主要的致死机制,并初步阐述了低温等离子体促进肿瘤细胞凋亡的分子机制.%Objective To explore the killing effect of dielectric barrier discharge (DBD) plasma on tumor cells and to analyze the DBD-induced apoptosis mechanism.Methods Thiazolyl blue tetrazolium bromide (MTT) assay method was used to detect the killing effect of low temperature plasma on the cytotoxicity of normal spleen leukocytes and acute promyelocytic leukemia cells (LT-12) at different doses.The changes of reactive oxygen species (ROS) level were measured after plasma treatment.The cell apoptosis rate was detected by Annexin V/PI double staining at different doses.The expression of apoptosis-related genes and proteins was detected by qRT-PCR and Western Blot.Results MTT results showed that the killing effect of plasma treatment was dose-dependent and time-dependent.The cell survival rate after 8 hours of treatment decreased from 98％ to 63％ with the dose increasing from 30 s to 240 s.The survival rate decreased from 78％ (2 h) to 39％ (24 h) after the treatment with a same dose (e.g.240 s).Annexin V/PI double staining results demonstrated that the plasma effect can induce apoptosis,and the apoptosis rate was not only positively correlated with the plasma dose,but also with the post-plasma time.The longer the post-plasma time,the higher was the apoptosis rate.The apoptotic rate of the 60 s dose treatment after 12 h was 48％ that increased to 55.3％ with the dose of 120 s.The production of reactive oxygen species (ROS) detected by flow cytometry also showed a time correlation of the plasma treatment.After the plasma treatment,the ROS level immediately increased to 1.24 times,and sharply increased to 5.39 times after 20 h post-plasma.The experimental results of qRT-PCR and Western blot showed that the expression of the genes and proteins of Caspase family and Bcl-2 family was very active at 8 to 12 h post-plasma treatment.Conclusions Low-temperature plasma can effectively kill tumor cells,and apoptosis is the main mechanism of death.The molecular mechanism of apoptosis of tumor cells induced by low temperature plasma was preliminary confirmed.