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目的观察放线菌素D(actinomycin D,ACTD)处理中国仓鼠成纤维靶细胞(the Chinese hamster fibroblast V79-C3cell line,V79细胞)获得的条件培养液(conditioned medium,CM)对V79旁观者细胞线粒体膜电位(Δψm)和胞质内细胞色素C(cytochrome C,Cyt C)含量的影响,以研究ACTD诱导旁观者效应发生的可能机制。方法用4.0mg/L ACTD处理V79靶细胞1h,磷酸盐缓冲液(phosphate buffer solution,PBS)漂洗3次,加入新鲜培养液开始计时,分别在第4、8、12和24h获取靶细胞条件培养液CM。不同时段CM培养正常V79细胞24h后,采用罗丹明123(Rhodamine 123,Rh123)荧光染色流式细胞仪法检测旁观者细胞的线粒体膜电位(Δψm);比色法测定胞质内Cyt C的含量。结果各CM处理组旁观者细胞Δψm均比对照组下降(P<0.01)。随着时段的延后,Δψm平均荧光强度在逐渐增加,24h又减少(P<0.01)。胞质内Cyt C含量以对照组最低,CM处理组胞质内Cyt C含量增加(P<0.01),4h最高,随后下降,24h又明显增加(P<0.01)。结论 ACTD诱导旁观者效应可能通过靶细胞的CM影响旁观者细胞线粒体膜电位下降和胞质内Cyt C含量增加,进而引起旁观者细胞凋亡。
Objective To observe the effect of actinomycin D (ACTD) treatment of conditioned medium (CM) obtained from Chinese hamster fibroblast V79-C3cell line (V79 cells) on the expression of mitochondria Membrane potential (Δψm) and cytochrome C (Cyt C) content in order to study the possible mechanism of bystander effect induced by ACTD. Methods V79 target cells were treated with 4.0 mg / L ACTD for 1 h. The cells were rinsed three times with phosphate buffer solution (PBS) and incubated with fresh medium. The target cells were harvested at 4, 8, 12 and 24 hours respectively Liquid CM. At different time periods, CMs were cultured in normal V79 cells for 24 h and the mitochondrial membrane potential (Δψm) of bystander cells was detected by Rhodamine 123 (Rh123) fluorescence staining. The content of Cyt C in cytoplasm . Results The Δψm of bystander cells in each CM treatment group was lower than that in the control group (P <0.01). With the lapse of time, the mean fluorescence intensity of Δψm gradually increased and decreased again after 24h (P <0.01). Cyt C content in the cytoplasm was the lowest in the control group. Cyt C content in the cytoplasm increased (P <0.01) in the CM treatment group, reached the peak at 4h, then decreased, and then increased significantly at 24h (P <0.01). Conclusion The bystander effect induced by ACTD may affect the bystander mitochondrial membrane potential and cytoplasmic Cyt C content increase by CM in target cells, which may cause bystander cell apoptosis.