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靶向c-Met的CAR-T细胞构建及其对人浆液性卵巢癌细胞SKOV-3的体外杀伤性

杜娜娜¹, 张艳君², 李艳秋¹, 张露², 安然³, 甄翔程⁴, 闵静婷⁴, 李正红^1,5,*

¹蚌埠医科大学基础医学院，蚌埠 233030；²蚌埠医科大学生命科学学院，蚌埠 233030；³蚌埠医科大学公共基础学院，蚌埠 233030；⁴蚌埠医科大学临床医学院，蚌埠 233030；⁵安徽省肿瘤演进及智能诊疗重点实验室，蚌埠 233030

摘要

本研究旨在构建靶向细胞间质上皮转换因子(c-mesenchymal-epithelial transition factor, c-Met)的第二代和第三代嵌合抗原受体T细胞(chimeric antigen receptor T cells, CAR-T)，观察其对人浆液性卵巢癌细胞SKOV-3 的体外杀伤作用。采用生物信息学分析MET基因在卵巢浆液性囊腺癌中的表达情况、MET基因表达与免疫细胞浸润丰度的相关性及其对卵巢浆液性囊腺癌组织功能的影响；免疫组织化学染色法检测浆液性卵巢癌组织以及癌旁组织中c-Met 的表达情况；流式细胞术检测SKOV-3 表面c-Met 蛋白的表达；通过慢病毒感染制备第二代和第三代c-Met CAR-T细胞，流式细胞术检测细胞亚群和感染效率。以CD19 CAR-T和活化T细胞为对照组，以c-Met阴性表达的人卵巢癌细胞A2780细胞设置Non target组，通过乳酸脱氢酶(lactate dehydrogenase, LDH)释放、ELISA、CCK-8 等功能实验探究第二代[c-Met CAR-T(2G)]和第三代[c-Met CAR-T(3G)]c-Met CAR-T对c-Met 阳性表达的SKOV-3 细胞的杀伤效果、细胞因子分泌和增殖能力。生物信息学分析与免疫组织化学染色结果显示，与正常组织相比，卵巢癌组织的MET基因表达显著上调，而在各病理阶段MET表达无明显差异，且MET基因表达与卵巢癌患者的种族、年龄均没有相关性。成功构建第二代和第三代c-Met CAR-T 细胞，且在慢病毒感染后c-MetCAR-T(2G)的CD8+ T 细胞比例有所上调，c-Met CAR-T(3G)细胞亚群无明显变化；LDH释放实验显示，第二代和第三代c-Met CAR-T对靶细胞SKOV-3的杀伤效率随效靶比提高而上升，效靶比为20:1时，c-Met CAR-T(2G)的杀伤效率达到(42.02 ±5.17)% (P < 0.05)，c-Met CAR-T(3G)的杀伤效率达到(51.40 ± 2.71)% (P < 0.05)；ELISA 结果显示，c-Met CAR-T 相较于CD19 CAR-T 和活化T 细胞能释放更多细胞因子(P < 0.05)，c-Met CAR-T(3G)的释放量高于c-Met CAR-T(2G) (P < 0.01)；CCK-8 结果显示，在48 h 后，c-Met CAR-T(2G)细胞数量显著多于c-Met CAR-T(3G) (P < 0.01)。以上结果提示，第二代和第三代c-Met CAR-T 均能靶向识别并杀伤c-Met 阳性的人浆液性卵巢癌细胞SKOV-3，两者的细胞毒性无明显差异，c-MetCAR-T(2G)的增殖能力更强，c-Met CAR-T(3G)释放细胞因子的水平更高。

关键词： 浆液性卵巢癌; c-Met; CAR-T; 生物信息学

c-Met-targeted chimeric antigen receptor T cells inhibit human serous ovarian cancer cell SKOV-3 in vitro

DU Na-Na¹, ZHANG Yan-Jun², LI Yan-Qiu¹, ZHANG Lu², AN Ran³, ZHEN Xiang-Cheng⁴, MIN Jing-Ting⁴, LI Zheng-Hong^1,5,*

¹School of Basic Medicine, Bengbu Medical University, Bengbu 233030, China；²School of Life Sciences, Bengbu Medical University, Bengbu 233030, China；³Public Basic College, Bengbu Medical University, Bengbu 233030, China；⁴Clinic Medical College, Bengbu Medical University, Bengbu 233030, China；⁵Anhui Province Key Laboratory of Tumor Evolution and Intelligent Diagnosis and Treatment, Bengbu 233030, China

Abstract

The study aimed to construct the second and third generation chimeric antigen receptor T cells (CAR-T) targeting the cmesenchymal- epithelial transition factor (c-Met) protein, and observe their killing effect on human serous ovarian cancer cell SKOV- 3. The expression of MET gene in ovarian serous cystadenocarcinoma, the correlation between MET gene expression and the abundance of immune cell infiltration, and the effect of MET gene expression on the tissue function of ovarian serous cystadenocarcinoma were analyzed by bioinformatics. The expression of c-Met in ovarian cancer tissues and adjacent tissues was detected by immunohistochemical staining. The second and third generation c-Met CAR-T cells, namely c-Met CAR-T(2G/3G), were prepared by lentivirus infection, and the cell subsets and infection efficiency were detected by flow cytometry. Using CD19 CAR-T and activated T cells as control groups and A2780 cells with c-Met negative expression as Non target groups, the kill efficiency on SKOV-3 cells with c-Met positive expression, cytokine release and cell proliferation of c-Met CAR-T(2G/3G) were explored by lactate dehydrogenase (LDH) release, ELISA and CCK-8 respectively. The results showed that MET gene expression was significantly up-regulated in ovarian cancer tissues compared with normal tissues, which was consistent with the immunohistochemistry results. However, in all pathological stages, there was no obvious difference in MET expression and no correlation between MET gene expression and the race and age of ovarian cancer patients. The second generation and third generation c-Met CAR-T cells were successfully constructed. After lentivirus infection, the proportion of CD8+ T cells in c-Met CAR-T(2G) was upregulated, while there was no significant change in the cell subsets of c-Met CAR-T(3G). The LDH release experiment showed that the kill efficiency of c-Met CAR-T(2G/3G) on SKOV-3 increased with the increase of effect-target ratio. When the effect-target ratio was 20:1, the kill efficiency of c-Met CAR-T(2G) reached (42.02 ± 5.17)% (P < 0.05), and the kill efficiency of c-Met CAR-T(3G) reached (51.40 ± 2.71)% (P < 0.05). ELISA results showed that c-Met CAR-T released more cytokine compared to CD19 CAR-T and activated T cells (P < 0.05). Moreover, the cytokine release of c-Met CAR-T(3G) was higher than c-Met CAR-T(2G) (P < 0.01). The CCK-8 results showed that after 48 h, the cell number of c- Met CAR-T(2G) was higher than that of c-Met CAR-T(3G) (P < 0.01). In conclusion, both the second and third generation c-Met CAR-T can target and kill c-Met-positive SKOV-3 cells, with no significant difference. c-Met CAR-T(2G) has stronger proliferative ability, and c-Met CAR-T(3G) releases more cytokines.

Key words: ovarian cancer; c-Met; CAR-T; bioinformatics

收稿日期：　　录用日期：

通讯作者：李正红　　E-mail:

DOI: 10.13294/j.aps.2025.0002

引用本文：

杜娜娜, 张艳君, 李艳秋, 张露, 安然, 甄翔程, 闵静婷, 李正红. 靶向c-Met的CAR-T细胞构建及其对人浆液性卵巢癌细胞SKOV-3的体外杀伤性[J]. 生理学报 2025; 77 (2): 241-254.

DU Na-Na, ZHANG Yan-Jun, LI Yan-Qiu, ZHANG Lu, AN Ran, ZHEN Xiang-Cheng, MIN Jing-Ting, LI Zheng-Hong. c-Met-targeted chimeric antigen receptor T cells inhibit human serous ovarian cancer cell SKOV-3 in vitro. Acta Physiol Sin 2025; 77 (2): 241-254 (in Chinese with English abstract).