摘　要：为研究探讨卡瑞利珠单抗联合顺铂通过调控线粒体氧化磷酸化（OXPHOS）、磷脂酰肌醇3激酶/蛋白激酶B（PI3K/AKT）信号通路对食管鳞癌细胞Eca-109凋亡的影响，并观察热休克蛋白A9（HSPA9）在其中的表达，本研究将试验分为空白对照组、顺铂组（100 μmol/L）和联合给药组［顺铂（100 μmol/L）+卡瑞利珠单抗（每次200 mg）］，运用Transwell迁移、划痕试验、细胞计数试剂盒-8（CCK-8）试验和克隆聚集技术分别观察卡瑞利珠单抗联合顺铂对食管鳞癌细胞Eca-109侵袭迁移能力、增殖活性的影响。利用逆转录聚合酶链反应（RT-PCR）检测HSPA9基因的表达；利用蛋白质印迹（Western blot）技术检测OXPHOS和PI3K/AKT通路蛋白的表达；利用流式细胞术检测线粒体活性氧（ROS）水平。Transwell迁移、划痕试验、CCK-8和克隆形成试验显示，联合给药组较顺铂组更能抑制食管鳞癌细胞Eca-109的活性、迁移和聚集；RT-PCR和Western blot检测结果显示，联合给药组与顺铂组和对照组相比较，HSPA9和OXPHOS的表达显著下降，并抑制PI3K/AKT发生磷酸化（P<0.01）；流式细胞术检测显示，与对照组和顺铂组相比较，顺铂+卡瑞利珠单抗组ROS水平明显增高，有显著性差异（P<0.05）。卡瑞利珠单抗联合顺铂相对于单独使用顺铂对食管鳞癌细胞Eca-109有明显抑制作用，其可能通过下调HSPA9和OXPHOS的表达，抑制PI3K/AKT信号通路发生磷酸化，调控氧化还原和线粒体稳态，诱导线粒体凋亡来发挥对食管鳞癌细胞Eca-109的抑制作用，为临床上食管鳞癌的精准干预和治疗提供了参考依据。
关键词：卡瑞利珠单抗；顺铂；食管鳞状细胞癌；OXPHOS；HSPA9
中图分类号：R735.1　　                  文献标志码：A　                  DOI：10.3969/j.issn.1007-7146.2025.03.010

Abstract: In order to study the effect of carrilizumab combined with cisplatin on apoptosis of esophageal squamous cell carcinoma cells Eca-109 by regulating mitochondrial oxidative phosphorylation (OXPHOS) and phosphatidylinositol 3 kinase/protein kinase B (PI3K/AKT) signaling pathway, and to observe the expression of heat shock protein A9 (HSPA9). In this study, the test was divided into blank control group, cisplatin group (100 μmol/L) and combined administration group which is cisplatin (100 μmol/L)+carrilizumab (200 mg/time). Transwell migration test, scratch test, cell counting kit 8 (CCK-8) test and clonal aggregation technique were used to observe the effects of carrellizumab combined with cisplatin on the invasion, migration and proliferation of esophageal squamous cell carcinoma cells Eca-109. The expression of HSPA9 gene was detected by RT-PCR. Western blot (WB) was used to detect the expression of OXPHOS and PI3K/AKT pathway proteins. Mitochondrial reactive oxygen species (ROS) were detected by flow cytometry. Transwell migration, scratch test, CCK-8 test and clonal formation test showed that the combined administration group inhibited the activity, migration and aggregation of Eca-109 cells more than the cisplatin group. Reverse transcription-polymerase chain reaction (RT-PCR) and WB detection results showed that compared with cisplatin group and control group, the expression of HSPA9 and OXPHOS in combined administration group significantly decreased, and the phosphorylation of PI3K/AKT was inhibited (P<0.01). Flow cytometry showed that compared with control group and cisplatin group, ROS levels in cisplatin+carrilizumab group significantly increased, and there was a significant difference (P<0.05). Compared with cisplatin alone, carrellizumab combined with cisplatin could significantly inhibit the expression of HSPA9 and OXPHOS in esophageal squamous cell cancer cells, inhibit the phosphorylation of PI3K/AKT signaling pathway, and regulate reoxidation and mitochondrial homeostasis. The inhibition of Eca-109 on esophageal squamous cell carcinoma by inducing mitochondrial apoptosis provides a reference for the precise intervention and treatment of esophageal squamous cell carcinoma in clinical practice.
Key words: carrilizumab; cisplatin; esophageal squamouscell carcinoma; OXPHOS; HSPA9
（Acta Laser Biology Sinica, 2025, 34(3): 274-281）