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目的对比研究mtDNA缺失以及再转入线粒体后细胞凋亡的变化。方法在成功构建ρ0SK-Hep1细胞和转线粒体细胞SK-Hep1 Cyb的基础上,采用Annexin V/PI双染色法检测细胞凋亡;流式细胞仪检测细胞内活性氧(ROS)和线粒体膜电位(ΔΨm);Western blot检测细胞Bcl-2、Bax表达水平;免疫荧光染色观测Bcl-2细胞内分布。结果SK-Hep1、ρ0SK-Hep1和SK-Hep1Cyb细胞凋亡率分别为(2.01±0.11)%、(0.37±0.08)%和(2.10±0.12)%。ρ0SK-Hep1对细胞凋亡有明显抗性(P<0.05)。ρ0SK-Hep1细胞内DCFDA荧光强度较SK-Hep1细胞显著增强(35.5与15.9,P<0.01);转入线粒体后,SK-Hep1Cyb细胞DCFDA荧光强度较ρ0SK-Hep1细胞明显下降(17.4与35.5,P<0.01)。ρ0SK-Hep1细胞MitoTracker Red荧光强度较SK-Hep1细胞显著减低(55.0与65.9,P<0.05);转入线粒体后,SK-Hep1Cyb细胞MitoTrack-er Red荧光强度与SK-Hep1细胞基本一致(67.4与65.9,P>0.05)。ρ0SK-Hep1细胞线粒体Bcl-2、Bax表达增多,Bcl-2/Bax比值增加(P<0.01)。SK-Hep1Cyb细胞线粒体Bcl-2/Bax值下降。结论mtDNA缺失肿瘤细胞对细胞凋亡有明显拮抗。Bcl-2线粒体转位、线粒体Bcl-2/Bax值增加、ROS产生增多可能参与细胞凋亡拮抗的形成。
Objective To compare the changes of mtDNA deletion and apoptosis after reintroduction into mitochondria. Methods The apoptosis of SKOV7 and SK-Hep1 cells was detected by Annexin V / PI double staining method. The levels of reactive oxygen species (ROS) and mitochondrial membrane potential ΔΨm). Western blot was used to detect the expression of Bcl-2 and Bax. The distribution of Bcl-2 cells was observed by immunofluorescence staining. Results The apoptosis rates of SK-Hep1, ρ0SK-Hep1 and SK-Hep1Cyb cells were (2.01 ± 0.11)%, (0.37 ± 0.08)% and (2.10 ± 0.12)%, respectively. ρ0SK-Hep1 had obvious resistance to apoptosis (P <0.05). The fluorescence intensity of DCFDA in ρ0SK-Hep1 cells was significantly higher than that in SK-Hep1 cells (35.5 and 15.9, P <0.01). After transfected into mitochondria, the fluorescence intensity of DCFDA in SK-Hep1Cyb cells was significantly lower than that of ρ0SK-Hep1 cells <0.01). The MitoTracker Red fluorescence intensity of ρ0SK-Hep1 cells was significantly lower than that of SK-Hep1 cells (55.0 and 65.9, P <0.05). The MitoTrack-er Red fluorescence intensity of SK-Hep1Cyb cells transfected into mitochondria was similar to that of SK-Hep1 cells 65.9, P> 0.05). The expression of Bcl-2, Bax and the ratio of Bcl-2 / Bax in ρ0SK-Hep1 mitochondria increased (P <0.01). The mitochondrial Bcl-2 / Bax decreased in SK-Hep1Cyb cells. Conclusion mtDNA deletion of tumor cells significantly inhibited apoptosis. Bcl-2 mitochondrial translocation, mitochondrial Bcl-2 / Bax value increased, ROS production may be involved in the formation of apoptosis antagonism.