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目的观察放线菌素D(actinomycin D,ACTD)处理V79靶细胞获得的条件培养液(conditioned medium,CM)对V79旁观者细胞ROS水平和细胞色素C(Cytochorome C,Cyt C)分布的影响,以研究ACTD诱导旁观者效应发生的可能机制。方法 4.0 mg/L ACTD处理V79靶细胞1 h,磷酸盐缓冲液(PBS)洗3次,加入新鲜培养液开始计时,分别在4、8、12和24 h获取靶细胞条件培养液CM。用不同时段CM培养正常V79细胞24 h后,流式细胞仪观察旁观者细胞ROS水平;比色法测定胞浆和线粒体细胞色素C含量。结果 CM处理组旁观者细胞ROS水平均高于对照组,随着时段的延后,ROS水平逐渐降低(P<0.01),24 h又增加(P<0.01)。对照组线粒体内Cyt C含量最高,在各CM处理组旁观者细胞线粒体Cyt C含量均有不同程度下降,4 h CM组含量最低,随着时段延后,线粒体Cyt C含量增加,但没达到对照组水平,24 h又趋下降。胞浆内Cyt C含量变化趋势与之相反,对照组Cyt C含量最低,CM处理组胞浆Cyt C含量增加,4 h最高随后下降,24 h又明显增加。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ROS的产生,并造成细胞色素C从线粒体释放而引起的。
Objective To observe the effects of conditioned media (CM) obtained from actinomycin D (ACTD) treatment on V79 target cells on the ROS level and cytochrome C (Cyt C) distribution in V79 bystander cells. To investigate the possible mechanisms by which ACTD induces bystander effects. Methods V79 target cells were treated with 4.0 mg / L ACTD for 1 h and PBS three times, respectively. Fresh medium was added for counting. The target cell conditioned medium CM was obtained at 4, 8, 12 and 24 h respectively. After cultured normal V79 cells for 24 h with different time periods, the ROS level of bystander cells was observed by flow cytometry. Cytochrome C content in cytoplasm and mitochondria was measured by colorimetric method. Results The level of ROS in bystander cells in CM treatment group was higher than that in control group. ROS levels decreased gradually (P <0.01) and increased again in 24 h (P <0.01). In the control group, the content of Cyt C in the mitochondria was the highest, while the content of Cyt C in the mitochondria of the CM treated groups decreased to different extents, and the content of Cyt C was the lowest in the 4 h. With the delay of time, the content of mitochondrial Cyt C increased but did not reach the control Group level, 24 h and tend to decline. On the contrary, Cyt C content in cytoplasm was the lowest, Cyt C content in control group was the lowest, cytoplasmic Cyt C content increased in CM treatment group, then decreased at 4 h and then increased significantly at 24 h. CONCLUSIONS: The bystander effect induced by ACTD may be caused by CM of target cells affecting the ROS production in bystander cells and the release of cytochrome C from mitochondria.