[c- met靶向嵌合抗原受体T细胞体外抑制人浆液性卵巢癌细胞SKOV-3]。

Q3 Medicine

生理学报 Pub Date : 2025-04-25 DOI:10.13294/j.aps.2025.0002

Na-Na DU, Yan-Jun Zhang, Yan-Qiu Li, Lu Zhang, Ran An, Xiang-Cheng Zhen, Jing-Ting Min, Zheng-Hong Li

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引用次数: 0

摘要

本研究旨在构建靶向c-间充质上皮转化因子（c-Met）蛋白的第二代和第三代嵌合抗原受体T细胞（CAR-T），观察其对人浆液性卵巢癌细胞SKOV-3的杀伤作用。应用生物信息学方法分析MET基因在卵巢浆液性囊腺癌中的表达、MET基因表达与免疫细胞浸润丰度的相关性以及MET基因表达对卵巢浆液性囊腺癌组织功能的影响。免疫组化染色检测c-Met在卵巢癌组织及癌旁组织中的表达。慢病毒感染制备第二代和第三代c-Met CAR-T细胞，即c-Met CAR-T细胞（2G/3G），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，c-Met阴性表达的A2780细胞为非靶组，分别通过乳酸脱氢酶（LDH）释放、ELISA和CCK-8检测c-Met CAR-T（2G/3G）对c-Met阳性表达的skv -3细胞的杀伤效率、细胞因子释放和细胞增殖情况。结果显示MET基因在卵巢癌组织中的表达较正常组织明显上调，与免疫组化结果一致。然而，在所有病理分期中，MET基因表达均无明显差异，且MET基因表达与卵巢癌患者的种族、年龄无相关性。成功构建第二代和第三代c-Met CAR-T细胞。慢病毒感染后，c-Met CAR-T细胞中CD8+ T细胞的比例（2G）上调，而c-Met CAR-T细胞亚群（3G）无明显变化。LDH释放实验表明，c-Met CAR-T（2G/3G）对SKOV-3的杀伤效率随着效靶比的增加而增加。效靶比为20:1时，c-Met CAR-T（2G）的杀伤效率为（42.02±5.17）% (P < 0.05), c-Met CAR-T（3G）的杀伤效率为（51.40±2.71）% （P < 0.05）。ELISA结果显示，与CD19 CAR-T和活化T细胞相比，c-Met CAR-T细胞释放的细胞因子更多（P < 0.05）。此外，c-Met CAR-T（3G）的细胞因子释放量高于c-Met CAR-T(2G) （P < 0.01）。CCK-8结果显示，48 h后，c-Met CAR-T（2G）细胞数高于c-Met CAR-T（3G）细胞数（P < 0.01）。综上所述，第二代和第三代c-Met CAR-T都可以靶向并杀死c-Met阳性的SKOV-3细胞，两者之间没有显著差异。c-Met CAR-T（2G）具有更强的增殖能力，c-Met CAR-T（3G）释放更多的细胞因子。

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

The study aimed to construct the second and third generation chimeric antigen receptor T cells (CAR-T) targeting the c-mesenchymal-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.

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来源期刊

生理学报 Medicine-Medicine (all)

CiteScore

1.20

自引率

0.00%

发文量

4820

期刊介绍： Acta Physiologica Sinica (APS) is sponsored by the Chinese Association for Physiological Sciences and Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences (CAS), and is published bimonthly by the Science Press, China. APS publishes original research articles in the field of physiology as well as research contributions from other biomedical disciplines and proceedings of conferences and symposia of physiological sciences. Besides “Original Research Articles”, the journal also provides columns as “Brief Review”, “Rapid Communication”, “Experimental Technique”, and “Letter to the Editor”. Articles are published in either Chinese or English according to authors’ submission.