诱导细胞mtDNA缺失以及再转入线粒体后对肿瘤细胞凋亡的影响

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ρ_0SK-Hepl细胞和转线粒体细胞SK-HeplCyb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(△ψm);Western blot检测细胞Bcl-2、Bax表达水平;免疫荧光染色观测Bel-2细胞内分布.结果 SK-Hepl、ρ~0SK-Hepl和sK-HeplCyb细胞凋亡率分别为(2.01±0.11)%、(0.37±0.08)%和(2.10±0.12)%.ρ~0SK-Hepl对细胞凋亡有明显抗性(P<0.05).ρ~0SK-Hepl细胞内DCFDA荧光强度较SK-Hepl细胞显著增强(35.5与15.9,P<0.01);转入线粒体后,SK-HeplCyb细胞DCFDA荧光强度较ρ~0SK-Hepl细胞明显下降(17.4与35.5,P<0.01).ρ~0SK-Hepl细胞MitoTracker Red荧光强度较SK-Hepl细胞显著减低(55.0与65.9,P<0.05);转入线粒体后,SK.HeplCyb细胞MitoTrack-er Red荧光强度与SK-Hepl细胞基本一致(67.4与65.9,P>0.05).ρ~0SK-Hepl细胞线粒体Bcl-2、Bax表达增多,Bcl-2/Bax比值增加(P<0.01).SK-HeplCyb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Bci-2/Bax值增加、ROS产生增多可能参与细胞凋亡拮抗的形成.

Apoptosis in mtDNA-depleted cells and its transmitochondrial cybrids

Objective To investigate cell apoptosis in mtDNA-depleted SK-Hepl cells and the transmito-chondrial cybrids on the basis of establishment of ρ~0 SK-Hepl (a mtDNA-depleted cell line derived from human hepatoma cell line SK-Hepl) and the transmitochondrial cybrids, SK-HeplCyb. Methods SK-Hepl cells,ρ~0 SK-Hepl cells, and the transmitochondrial cybrids were cultured in high-glucose DMEM. Apoptosis was de-termined by Annexin V/PI assay. Flow cytometry (FCM) and laser scan confoeal microscopy were used to de-tect reactive oxygen species (ROS) and mitochondrial membrane potential (△ψm) respectively. Western blot analysis was applied to detect Bcl-2 and Bax expressions in the above 3 groups of cells. Distribution of Bcl-2 in the 3 groups was assessed by immunofluorescence analysis. Results Apoptotic ratio of SK-Hepl, ρ~0 SK-Hepl and SK-HeplCyb cells were (2.01±0.11)%, (0.37±0.08)% and (2.10±0.12)%, respectively.ρ~0 SK-Hepl cells were more counteractive against apoptosis than the other 2 cells (P <0.05). Fluorescence in-tensity of ROS in ρ~0 SK-Hepl cells was significantly higher than that of the parent cells (35.5 vs 15.6, P <0.01), but the △ψm was comparatively lower (55.0 vs 65.9, P < 0.05), meanwhile the ratio of Bcl-2/Bax was elevated. After transmitochondria to ρ~0 SK-Hepl cells, the SK-Hepl Cyb cells had all the above reversed. Conclusion mtDNA-depleted cells are more counteractive against apoptosis, and it might be related to the el-evated mitochondrial Bcl-2/Bax and ROS.