目的 探讨血糖波动对hCMEC/D3人脑微血管内皮细胞血脑屏障功能铜稳态的潜在影响。 方法 本研究以常规培养hCMEC/D3细胞，分为对照组（对照组，glucose 25 mmol/L培养4 d），葡萄糖浓度波动组（fluctuating glucose， FG 组，glucose 25 mmol/L培养8 h，glucose 55 mmol/L培养8 h，共培养4 d）；CCK-8法检测各组细胞的活力变化；用光学显微镜观察各组细胞的形态；通过Transwell装置上室中构建细胞单层膜，每天监测细胞跨内皮细胞电阻值变化；通过苯酚红检测单层细胞的通透性；检测各组细胞内铜离子含量以及细胞内ROS水平；qPCR检测铜转运蛋白1（SLC31A1）、铜转运ATP酶β链（ATP7B）、细胞色素c氧化酶铜伴侣蛋白17（COX17）、抗氧化剂1铜伴侣蛋白（ATOX1）mRNA表达情况；Western blot检测SLC31A1、ATP7B、COX17、ATOX1、闭合蛋白-1（ZO-1）、闭锁蛋白（Occludin）、紧密连接蛋白（Claudin-1）表达情况。 结果 血糖波动组的细胞活力显著低于对照组（P < 0.05）；对照组细胞生长状态良好，交织成致密网状，突触间相互连接；血糖波动组细胞生长受抑，数量明显少于对照组，细胞间突触的连接减少；血糖波动组跨内皮细胞电阻值（TEER值）低于对照组（P < 0.05），苯酚红通透性实验结果表明血糖波动组单层细胞完整性被破坏，通透性升高（P < 0.05）；铜离子检测试剂盒（P < 0.01）和铜离子荧光探针（P < 0.05）均显示血糖波动组细胞内铜离子含量降低，血糖波动组细胞内ROS水平在第4天明显升高（P < 0.05）；qPCR结果显示与对照组相比，血糖波动组ATOX1、COX17、SLC31A1表达降低（P < 0.05），ATP7B（P < 0.05）表达增加；Western blot结果显示与对照组相比，血糖波动组ZO-1、Occludin、Claudin-1表达降低（P < 0.001）；SLC31A1、ATOX1、COX17表达降低（P < 0.05），ATP7B表达增加（P < 0.05）；免疫荧光检测SLC31A1、ATP7B表达结果显示，与对照组相比，SLC31A1表达降低（P < 0.05），ATP7B表达上升（P < 0.001）。 结论 血糖波动可能通过诱导hCMEC/D3细胞屏障结构受损、增加细胞通透性以及诱发ROS累积等机制，破坏血脑屏障的结构与功能。这一过程可能与细胞内铜稳态失衡有关。

Objective To investigate the potential effects of glycemic fluctuation on copper homeostasis and blood–brain barrier （BBB） function in hCMEC/D3 human brain microvascular endothelial cells. Methods hCMEC/D3 cells were conventionally cultured and divided into a control group （glucose 25 mmol/L for 4 days） and a fluctuating glucose group （FG group： glucose 25 mmol/L for 8 h followed by glucose 55 mmol/L for 8 h， repeatedly for 4 days）. Cell viability was assessed using the CCK-8 assay， and cell morphology was observed under an light microscope. A Transwell system was used to construct a monolayer cell model， and transendothelial electrical resistance （TEER） was measured daily. Monolayer permeability was evaluated using the phenol red assay. Intracellular copper levels and reactive oxygen species （ROS） levels were detected. The mRNA expression levels of copper transporter 1 （SLC31A1）， copper-transporting ATPase β chain （ATP7B）， cytochrome c oxidase copper chaperone 17 （COX17）， and antioxidant 1 copper chaperone （ATOX1） were determined by qPCR. The protein expression levels of SLC31A1， ATP7B， COX17， ATOX1， zonula occludens-1 （ZO-1）， occludin， and Claudin-1 were analyzed by Western blot. Results Cell viability in the FG group was significantly lower than that in the control group （P < 0.05）. Cells in the control group exhibited robust growth and formed a dense network with extensive intercellular connections， whereas cell growth in the FG group was inhibited， with markedly reduced cell numbers and diminished intercellular contacts. The TEER value in the FG group was significantly lower than that in the control group （P < 0.05）， and the phenol red permeability assay revealed increased permeability and disrupted monolayer integrity in the FG group （P < 0.001）. Both the copper ion detection kit and copper-specific fluorescent probe showed a significant reduction in intracellular copper levels in the FG group. Intracellular ROS levels were markedly elevated on day 4 in the FG group （P < 0.05）. qPCR results demonstrated that， compared with the control group， the mRNA expression of ATOX1， COX17， and SLC31A1 was significantly decreased， whereas ATP7B expression was significantly increased （P < 0.05）. Western blot analysis showed that the protein expression of ZO-1， occludin， and Claudin-1 was significantly reduced in the FG group （P < 0.05）. The protein expression of SLC31A1 （P < 0.05）， ATOX1， and COX17 was decreased， while ATP7B expression was increased （P < 0.05）. Immunofluorescence analysis further confirmed that， compared with the control group， SLC31A1 expression was reduced and ATP7B expression was elevated in the FG group（P < 0.05）. Conclusion Glycemic fluctuation may impair the structure and function of the BBB in hCMEC/D3 cells by inducing damage to barrier-related structures， increasing cellular permeability， and promoting ROS accumulation. This pathological process may be closely associated with intracellular copper homeostasis imbalance， providing new insights into the mechanisms underlying BBB dysfunction in conditions characterized by glycemic instability.