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用不同剂量(0,0.25,0.5,1.0,2.0,4.0 mg/L)的放线菌素D(actinomycin D,ActD)诱导Vero细胞凋亡不同时间(0,12,24,36,48,60 h)。MTT法和流式细胞术的结果显示,以未处理的细胞作为对照组,各剂量ActD处理Vero细胞不同时间后,细胞的活力均下降,凋亡率均升高,且呈剂量和时间依赖效应。当ActD浓度为1 mg/L、作用时间为36 h,细胞的存活率为(0.55±0.01),凋亡率为(34.83±1.13)%。Gimesa染色、Hoechst33258染色表明,经ActD(1 mg/L)诱导36 h后Vero细胞形态学发生明显改变,出现膜小泡和凋亡小体形成等凋亡细胞特征;Real-time PCR结果显示,诱导组Bax/Bcl-2 mRNA相对含量明显大于正常对照组,且差异有统计学意义;分光光度法结果表明,与正常对照组相比较,凋亡诱导组caspase-3和caspase-8活性明显升高且差异具有统计学意义,caspase-9活性与对照组相比较,差异无统计学意义。用1 mg/L ActD诱导Vero细胞36 h时,细胞存活率和凋亡率都很高,可作为ActD诱导Vero细胞的最佳剂量。在该剂量及作用时间下,caspase-3和caspase-8活性明显升高,表明ActD诱导Vero细胞凋亡是通过caspase-8相关的途径。该实验成功建立了ActD诱导非洲绿猴肾细胞凋亡模型,将有助于进一步探讨目的基因在Vero细胞凋亡作用的的分子机制。
Vero cells were induced to apoptosis at different times (0, 12, 24, 36, 48, 60, 60, 60, h). The results of MTT assay and flow cytometry showed that untreated cells served as the control group. After treated with ActD for different time, the viability of Vero cells decreased and the apoptosis rate increased in a dose-and time-dependent manner . When the ActD concentration was 1 mg / L and the action time was 36 h, the cell survival rate was (0.55 ± 0.01) and the apoptosis rate was (34.83 ± 1.13)%. The results of Giemsa staining and Hoechst 33258 staining showed that morphological changes of Vero cells were induced by ActD (1 mg / L) for 36 h, and apoptotic cells such as membrane vesicles and apoptotic bodies appeared. Real-time PCR results showed that, The relative content of Bax / Bcl-2 mRNA in induction group was significantly higher than that in normal control group, and the difference was statistically significant. The results of spectrophotometry showed that compared with normal control group, the activity of caspase-3 and caspase-8 were significantly increased High and the difference was statistically significant, caspase-9 activity compared with the control group, the difference was not statistically significant. When Vero cells were treated with 1 mg / L ActD for 36 h, the cell viability and apoptosis rate were very high, which could be the optimal dose for Vero cells induced by ActD. At this dose and time, caspase-3 and caspase-8 activities were significantly increased, indicating that ActD induced apoptosis in Vero cells through a caspase-8-related pathway. The experiment successfully established ActD induced apoptosis model of African green monkey renal cell apoptosis, which will be helpful to further explore the molecular mechanism of the target gene in Vero cell apoptosis.