为了研究二甲双胍抑制结直肠癌干细胞（CSCs）自我更新的机制，我们通过荧光活化细胞分选（FACS）从Wnt感应物转染的结直肠癌患者源性器官样细胞瘤（PDOs）中分离了CSCs，并进行了二甲双胍处理。使用球形形成、集落形成和浓度稀释实验评估细胞自我更新的变化。通过定量逆转录聚合酶链反应（qRT-PCR）检测与干细胞特性和分化相关的基因和Wnt靶标基因的mRNA表达。使用流式细胞术评估CSCs中的Wnt活性。二甲双胍处理后，使用海马分析评估细胞氧耗速率（OCR）和细胞外酸化速率（ECAR）。使用TMRE染色检测线粒体膜电位水平，并使用MitoSOX染色检测活性氧（ROS）水平。使用半乳糖（10 mmol/L）、二甲双胍（10 μmol/L）、NAC（5 mmol/L）和半乳糖+二甲双胍对CSCs中的ROS水平进行调节，并使用球形形成实验和流式细胞术评估自我更新能力和Wnt活性的变化。通过流式细胞术分析酵母NADH脱氢酶NDI1的慢病毒转染对CSCs中TMRE染色、MitoSOX染色和Wnt活性的影响。二甲双胍显著降低CSCs形成球形、集落和异种移植物的能力，并减少细胞中的Wnt活性（P < 0.01）。与对照组相比，二甲双胍处理组干细胞特性相关基因和Wnt靶标基因的mRNA水平显著降低，分化相关基因的mRNA水平显著增加（P < 0.05），细胞的OCR、TMRE和ROS水平显著降低，而ECAR增加（P < 0.001）。半乳糖显著增加细胞的球形形成能力、ROS水平和Wnt活性，而这些效应被二甲双胍显著抑制（P < 0.05）。CSCs转染NDI1显著减弱了二甲双胍对CSCs比例和Wnt信号通路活性的抑制作用。二甲双胍通过抑制线粒体复合物Ⅰ来降低线粒体氧化磷酸化和ROS水平，从而抑制Wnt信号通路，减少结直肠CSCs的自我更新能力。

To investigate the mechanism of metformin for inhibiting self-renewal of colorectal cancer stem cells (CSCs).CSCs were sorted from Wnt reporter- transfected colorectal cancer patient-derived organoids (PDOs) by fluorescence-activated cell sorting (FACS) and treated with metformin. The changes in self-renewal of the cells were assessed using sphere formation, colony formation and limiting dilution assays. The mRNA expressions of genes related with stemness and differentiation and Wnt target genes was detected by qRT-PCR. Wnt activity was assessed using flow cytometry in the CSCs. Seahorse analysis was used to evaluate cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) after metformin treatment. Mitochondrial membrane potential levels were detected with TMRE staining, and reactive oxygen species (ROS) levels were detected using MitoSOX staining. Galactose (10 mmol/L), metformin (10 μmol/L), NAC (5 mmol/L), and galactose+metformin were used to modulate ROS levels in the CSCs, and sphere-formation assay and flow cytometry were used to assess the changes in self- renewal capacity and Wnt activity. The effect of lentiviral transfection of yeast NADH dehydrogenase NDI1 on TMRE staining, MitoSOX staining and Wnt activity in the CSCs were analyzed with flow cytometry.Metformin significantly decreased the capacities of CSCs to form spheres, colonies and xenografts and reduced Wnt activity in the cells (P < 0.01). The mRNA levels of stemness-related genes and Wnt target genes decreased significantly while those of differentiation-related genes increased in metformin-treated CSCs (P < 0.05), which also showed significantly decreased OCR, TMRE and ROS levels with enhanced ECAR (P < 0.001). Galactose significantly increased sphereforming capacity, ROS levels and Wnt activity of the cells, and these effects were significantly inhibited by metformin (P < 0.05). Transfection of the CSCs with NDI1 significantly attenuated the inhibitory effects of metformin on proportion of CSCs and Wnt signaling pathway activity.Metformin reduces mitochondrial oxidative phosphorylation and ROS levels by inhibiting mitochondrial complex Ⅰ, thereby suppressing Wnt signaling pathway to reduce selfrenewal ability of colorectal CSCs.