温石棉对人支气管上皮细胞BEAS-2B凋亡的影响

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／cm^2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／cm^2温石棉、5μg／cm^2 SiO2、20μg／cm^2 WO1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／cm^2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。

Effects of Chrysotile Fibers on Apoptosis of Human Bronchial Epithelial Cell Line(BEAS-2B)in vitro

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. Mothods ] BEAS-2B cell line was cultured together with 5, 10, 20, 40, 100, and 200μg/cm^2 chrysotile fibers separately for 24, 48, and 72h, using 5μg/cm^2 quartz（ SiO2 ）as postive control and 20μg/cm^2wollastonite（ WO1 ） as negtive control. The viability of BEAS-2B cells was detected by MTr 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 ]10μg/cm^2 ehrysotile 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 72h 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 ehrysotile fibers.