Objectives: To observe the mitochondrial morphology of normal and triple-negative breast cancer cells, extract mitochondria from normal cells, and investigate the effects of mitochondrial transplantation on proliferation, apoptosis, and stemness of triple-negative breast cancer cells. Methods: The morphology of mitochondria was observed by transmission electron microscope. Mitochondria were extracted by mitochondrial extraction kit, mitochondrial protein was identified by western blot, and mitochondrial activity was detected by mitochondrial membrane potential detection kit. MitoTracker Green or MitoTracker Deep Red fluorescent probes were used to label the mitochondria of living cells, and the degree of mitochondria entering LTT cells was observed by confocal laser microscopy at 12, 24, and 96 hours. The effects of mitochondrial transplantation on proliferation, apoptosis, and stemness of breast cancer cells were examined by CCK8, colony formation assay, flow cytometry, and sphere formation assay after 24 hours of mitochondrial transplantation. Results: The mitochondria of normal cells were rod-shaped or elongated, while the mitochondria of triple-negative breast cancer cells were swollen and vacuolated. Western blot results showed that cytochrome c oxidase subunit I (MT-CO1) protein encoded by mitochondria was present in the isolated mitochondria. The content of heat shock protein 60 (HSP60) was higher in mitochondria than that in cytoplasm. The result of the multi-mode microplate reader showed that the content of mitochondrial J-aggregates/monomer was 1.67±0.06, which was significantly higher than 0.35±0.04 of the control group (P＜0.001). Exogenous mitochondria were observed in LTT cells at 12, 24, and 96 hours after mitochondrial transplantation. The results of the CCK8 experiment showed that OD450 of LTT cells was 0.27±0.13 after 48 hours transplantation, which was lower than 0.62±0.36 of the control group (P=0.023). The OD450 of MDA-MB-468 cells was 0.30±0.03, which was lower than 0.65±0.10 of the control group (P=0.004). After 120 hours of mitochondrial transplantation, OD450 in both groups was still significantly lower than that in the control group (P＜0.01). The number of clones formed by mitochondrial transplantation of LTT cells was 21.33±7.31, which was lower than 35.22±13.59 of the control group (P=0.016). Flow cytometry showed that the early apoptosis rate of LTT cells was (30.07±2.15)% after 24 hours of mitochondrial transplantation, which was higher than 2.07±1.58 of the control group (P＜0.001). The proportion of early apoptosis in MDA-MB-468 cells was 24.47%±5.22%, which was higher than (7.83±2.06)% in the control group (P=0.007). In addition, the number of mitochondria transplanted LTT cells into the cell sphere was 46.25±5.40, which was significantly lower than 62.58±6.43 of the control group (P＜0.001). Conclusion: Normal mitochondria can enter triple-negative breast cancer cells by co-culture, inhibit the proliferation and stemness of triple-negative breast cancer cells, and promote the apoptosis of triple-negative breast cancer cells.
目的： 观察正常细胞和三阴性乳腺癌细胞线粒体形态，提取正常细胞线粒体，探讨线粒体移植对三阴性乳腺癌细胞增殖、凋亡和干性的影响。 方法： 使用透射电子显微镜观察细胞线粒体形态，用线粒体提取试剂盒提取线粒体，Western blot鉴定线粒体蛋白，线粒体膜电位检测试剂盒检测线粒体活性。用MitoTracker Green或MitoTracker Deep Red荧光探针标记活细胞线粒体，在12、24、96 h用激光共聚焦显微镜观察线粒体进入三阴性乳腺癌LTT细胞的程度；线粒体移植24 h后利用细胞计数试剂盒8（CCK-8）、平板克隆、流式细胞术、细胞成球实验检测线粒体移植对三阴性乳腺癌细胞增殖、凋亡和干性的影响。 结果： 透射电子显微镜观察到正常细胞线粒体为长杆状、棒状，三阴性乳腺癌细胞线粒体肿胀及基质空泡样。Western blot结果显示，线粒体编码的细胞色素C氧化酶I蛋白存在于提取的线粒体中，线粒体中热休克蛋白60的含量高于细胞质。多模式微孔板检测仪结果显示，提取的线粒体JC-1聚合物/单体为1.67±0.06，高于对照组（0.35±0.04，P＜0.001）。线粒体移植后，12、24、96 h在LTT细胞中可观察到外源性线粒体。CCK-8实验结果显示，在移植48 h后，LTT细胞A值为0.27±0.13，低于对照组（0.62±0.36，P=0.023）。线粒体移植120 h时，LTT细胞A值仍低于对照组（均P＜0.01）。经线粒体移植处理的LTT细胞形成的克隆细胞数为（21.33±7.31）个，低于对照组（35.22±13.59，P=0.016）。流式细胞术结果显示，线粒体移植24 h后，LTT细胞早期凋亡比例为（30.07±2.15）%，高于对照组［（2.07±1.58）%，P＜0.001］，在MDA-MB-468细胞中，早期凋亡比例是（24.47±5.22）%，高于对照组［（7.83±2.06）%，P=0.007］。此外，细胞成球实验中，线粒体移植的LTT细胞成球数量为（46.25±5.40）个，低于对照组［（62.58±6.43）个，P＜0.001］。 结论： 正常线粒体可以通过共培养方式进入到三阴性乳腺癌细胞中，抑制三阴性乳腺癌细胞增殖和干性，促进三阴性乳腺癌细胞凋亡。.