目的 合成了具有荧光定位可视化功能的新型过氧麦角甾醇衍生物[过氧麦角甾醇-花菁染料Cy5（EP-Cy5）]，揭示其对乳腺癌MCF-7细胞的作用机制。方法 通过酯化反应将EP与花菁染料Cy5通过酯化反应得到EP-Cy5。采用MTT法检测不同浓度EP-Cy5处理MCF-7细胞24、48、72 h后的增殖抑制效果。利用激光共聚焦显微镜考察细胞摄取及共定位：以红色荧光标记的EP-Cy5染色细胞，同时分别用绿色荧光标记细胞核、脂滴、高尔基体、溶酶体、内质网和线粒体等细胞器进行共染色分析；使用斑马鱼模型评估EP-Cy5的活体成像性能。为深入探究EP-Cy5抑制MCF-7细胞的具体机制，采用流式细胞术检测EP-Cy5对MCF-7细胞的凋亡率、活性氧（ROS）水平和线粒体膜电位变化（JC-1）的影响；通过Western blotting法检测EP-Cy5对线粒体凋亡相关蛋白的表达。结果 EP-Cy5对MCF-7细胞的半数抑制浓度（IC₅₀）为4.48 μmol/L，是母体药物EP（IC₅₀＝19.02 μmol/L）的4.25倍。EP-Cy5的荧光强度呈时间–浓度相关性增长；与线粒体荧光探针高度共定位；细胞总凋亡率、ROS水平显著升高，线粒体膜电位显著下降（P＜0.01、0.001）；Western blotting结果表明，B淋巴细胞瘤-2基因（Bcl-2）蛋白表达显著下降，而Bcl-2相关X蛋白（Bax）、细胞色素C（Cyt-C）、剪切型半胱天冬酶-9（cleaved Caspase-9）及剪切型半胱天冬酶-7（cleaved Caspase-7）蛋白表达显著升高（P＜0.01、0.001）。结论 化合物EP-Cy5主要富集于乳腺癌MCF-7细胞线粒体，可升高ROS水平并显著降低线粒体膜电位，调节凋亡相关蛋白的表达。

Objective This study designed and synthesized a novel ergosterol peroxide derivative EP-Cy5 with fluorescence localization and visualization function, aiming to reveal its mechanism of action on breast cancer MCF-7 cells. Methods EP-Cy5 was obtained by conjugating EP with the cyanine dye Cy5 via an esterification reaction. The MTT assay was used to evaluate the antiproliferative effects of various concentrations of EP-Cy5 on MCF-7 cells after 24, 48, and 72 hours of treatment. Cellular uptake and co-localization were examined using confocal laser scanning microscopy: cells were stained with red fluorescent EP-Cy5, while simultaneously co-stained with green fluorescent probes targeting the nucleus, lipid droplets, Golgi apparatus, lysosomes, endoplasmic reticulum, and mitochondria. In vivo imaging performance of EP-Cy5 was assessed using a zebrafish model. To deeply explore the specific mechanism by which EP-Cy5 inhibits MCF-7 cells, flow cytometry was used to detect the effects of EP-Cy5 on the apoptosis rate, ROS level and mitochondrial membrane potential change (JC-1) of MCF-7 cells. The expression of mitochondrial apoptosis-related proteins was detected by Western blotting. Results The antiproliferative activity (IC₅₀) of EP-Cy5 against MCF-7 cells was 4.48 μmol/L, which was 4.25 times that of the parent drug EP (IC₅₀= 19.02 μmol/L). The fluorescence intensity of EP-Cy5 increased in a time-concentration dependent manner. Highly co-localized with mitochondrial fluorescent probes, the total apoptosis rate of cells and ROS were increased (P < 0.01, 0.001), and the mitochondrial membrane potential decreased significantly. Western blotting indicated that the expression of the Bcl-2 were decreased, the protein expressions of Bax, Cyt-c, cleaved Caspase-9, and cleaved Caspase-7 were increased (P < 0.01, 0.001). Conclusion In vitro experiments showed that compound EP-Cy5 mainly accumulated in the mitochondria of breast cancer MCF-7 cells. It could increase the level of ROS and significantly reduce the mitochondrial membrane potential, and regulate the expression of apoptosis-related proteins.

R979.1

齐齐哈尔市科学技术局联合引导项目（LSFGG-2024111）