【摘要】 [目的]研究温石棉对永生化人支气管上皮细胞(BEAS-2B)凋亡的影响,比较单细胞凝胶电泳试验(SCGE)与流式细胞术(FCM)检测细胞凋亡结果的异同。[方法]选用BEAS-2B细胞为受试物,以5、10、20、40、100、200μg/cm2不同剂量的温石棉,分别刺激BEAS-2B细胞24、48、72h,阳性对照用石英(SiO2),阴性对照用硅灰石(WO1),用四甲基偶氮唑盐(MTT)比色法检测温石棉对BEAS-2B活力的影响。用终剂量10μg/cm2温石棉、5μg/cm2SiO2、20μg/cm2WO1分别刺激细胞24、48和72h后,采用SCGE和FCM检测温石棉对BEAS-2B凋亡指数和凋亡率的影响。采用逆转录聚合酶链反应(RT-PCR)技术检测凋亡相关半胱氨酸蛋白酶-3基因caspase-3、caspase-9的表达。[结果]10μg/cm2温石棉可诱导BEAS-2B细胞凋亡,呈时间-效应关系(P<0.05),采用SCGE和FCM方法检测细胞凋亡率结果一致。温石棉刺激BEAS-2B细胞24、48、72h后凋亡指数为11.7%、21.8%和34.5%,凋亡率为9.6%、17.9%和31.2%;温石棉诱导细胞caspase-3、caspase-9表达水平随时间增加而增加。[结论]温石棉可诱导BEAS-2B凋亡;SCGE和FCM均可以灵敏、快捷地检测温石棉对BEAS-2B的凋亡作用。更多还原

【Abstract】 [Objective] To investigate the influence on apoptosis of human bronchial epithelial cell line(BEAS-2B)induced by chrysotile fibers and to compare the methods of single cell gel electrophoresis assay(SCGE)with flow cytometry analysis(FCM)for detecting apoptosis.[Methods] BEAS-2B cell line was cultured together with 5,10,20,40,100,and 200 μg/cm2 chrysotile fibers separately for 24,48,and 72 h,using 5 μg/cm2 quartz(SiO2)as postive control and 20 μg/cm2 wollastonite(WO1)as negtive control.The viability of BEAS-2B cells was detected by MTT assay,and the apoptotic effects were observed by SCGE and FCM.The mRNA levels of caspases-3 and caspases-9 were determined using reverse transtription polymerase chain reaction(RT-PCR).[Results] 10 μg/cm2 chrysotile fibers could induce a time-dependent apoptosis in BEAS-2B cells.The results of SCGE and FCM were consisitent.The apoptosis indices were 11.7%,21.8% and 34.5% and apoptosis rate were 9.6%,17.9% and 31.2% by stimulating BEAS-2B for 24,48,and 72 h respectively.The mRNA level of caspases-3 and caspases-9 increased with time in BEAS-2B cells treated with chrysotile fibers.[Conclusion] Chrysotile fibers could induce apoptosis of BEAS-2B cells.Both SCGE and FCM could be two good methods to detect apoptotic effects induced by chrysotile fibers.更多还原