靶向c-Met/CD47 CAR-T细胞对卵巢癌细胞的作用

doi:10.19803/j.1672-8629.20240339

摘要/Abstract

摘要： 目的体外研究敲减间充质上皮转化因子（c-Met）的表达对卵巢癌细胞增殖、侵袭、迁移及凋亡等影响,制备靶向c-Met的嵌合抗原受体T（CAR-T）并观察对卵巢癌细胞的杀伤效能。方法采用实时荧光定量聚合酶链式反应（RT-qPCR）、蛋白质印迹（WB）检测卵巢癌细胞系c-Met的表达情况;转染敲减c-Met,用细胞计数试剂盒-8(CCK-8)、细胞克隆检测细胞增殖情况;细胞侵袭(Transwell)、细胞划痕检测细胞侵袭、迁移能力;流式细胞术检测细胞周期、凋亡情况及慢病毒感染细胞的阳性率;CCK-8检测CAR-T细胞在靶细胞刺激下的增殖能力;乳酸脱氢酶(LDH)检测细胞的杀伤率;酶联免疫吸附试验(ELISA)检测干扰素-γ(IFN-γ)和白介素-2(IL-2)的释放情况。建立裸鼠皮下移植瘤模型,注射T、CAR-T、c-Met CAR-T、c-Met/CD47 CAR-T效应细胞组,测肿瘤重量,苏木精-伊红(HE)染色、免疫组化,研究CAR-T细胞对卵巢癌的治疗作用。结果显示SKOV3细胞系c-Met表达量高;与对照组相比,敲减c-Met组细胞增殖、侵袭、迁移能力降低及凋亡率增加;G1、S期降低,G2期增加;流式细胞术检测显示CAR-T、c-Met CAR-T、c-Met/CD47 CAR-T细胞的感染效率分别为97.42%、97.39%、97.35%;CCK-8检测显示c-Met /CD47 CAR-T细胞对靶细胞的增殖潜力显著增强;LDH检测显示c-Met/CD47 CAR-T细胞对靶细胞的杀伤率显著增加;ELISA检测表明SKOV3诱导c-Met/CD47 CAR-T细胞释放更多的IFN-γ和IL-2;皮下注射SKOV3细胞时c-Met/CD47 CAR-T细胞组裸鼠移植瘤重量小于T、CAR-T、c-Met CAR-T细胞组;HE结果显示各细胞治疗组中组织器官均未见生理结构破坏;免疫组化结果显示c-Met/CD47 CAR-T细胞组中c-Met阳性着色较弱。结论 c-Met/CD47可作为CAR-T细胞治疗卵巢癌的分子靶点,并能够对c-Met表达越高的卵巢癌细胞靶向识别、杀伤能力越强,同时更能提高增殖能力和细胞因子释放水平。

关键词: 卵巢癌, 间充质上皮转化因子, 间充质上皮转化因子/分化簇, 嵌合抗原受体T, 靶向, 细胞

Abstract: Objective To investigate the effect of knocking down the expression of mesenchymal-epithelial transition factor (c-Met) on proliferation, invasion, migration and apoptosis of ovarian cancer cells in vitro, prepare chimeric antigen receptor T (CAR-T) targeting c-Met and observe its ability to kill ovarian cancer cells. Methods The expression of c-Met in three ovarian cancer cell lines was detected by real-time fluorescence quantitative polymerase chain reaction and Western blotting. The c-Met was knocked down by transfection, and cell proliferation was detected by cell counting kit-8 (CCK-8) and cell cloning. Cell invasion and cell scratch were used to detect cell invasion and migration ability. The cell cycle, apoptosis and the positive rate of lentivirus infected cells were detected by flow cytometry. CCK-8 was used to detect the proliferation of CAR-T cells stimulated by target cells. Lactate dehydrogenase assay was used to detect the killing rate of cells while the release of interferon-γ (IFN-γ) and interleukin-2 (IL-2) was detected by enzyme-linked immunosorbent assay. A subcutaneous xenograft model of nude mice was established before T, CAR-T, c-Met CAR-T, c-Met/CD47 CAR-T effector cell groups were injected. The tumor weight was measured. Hematoxylin-eosin staining and immunohistochemistry were used to study the therapeutic effect of CAR-T cells against ovarian cancer. Results The expression of c-Met in SKOV3 cell line was high. Compared with the control group, the cell proliferation, invasion, migration ability of the c-Met knockdown group decreased while the apoptosis rate increased. G1 and S phase decreased while G2 phase increased. Flow cytometry showed that the infection efficiency of CAR-T, c-Met CAR-T and c-Met/CD47 CAR-T cells was 97.42%, 97.39% and 97.35%, respectively. CCK-8 assay indicated that the proliferation potential of c-Met/CD47 CAR-T cells to target cells was significantly enhanced. LDH assay suggested that the killing rate of c-Met/CD47 CAR-T cells to target cells was significantly increased. ELISA showed that SKOV3 induced c-Met/CD47 CAR-T cells to release more IFN-γ and IL-2. The weight of transplanted tumor in the c-Met/CD47 CAR-T cell group was lighter than in T, CAR-T and c-Met CAR-T cell groups when SKOV3 cells were injected subcutaneously. HE results showed that there was no physiological structure damage in the tissues and organs of each cell treatment group. Conclusion c-Met/CD47 can be used as a molecular target for CAR-T cell therapy for ovarian cancer, which can target and recognize ovarian cancer cells with higher c-Met expression. The stronger the killing ability, the better the proliferation ability and cytokine release level.

Key words: ovarian cancer, mesenchymal-epithelial transition factor, mesenchymal-epithelial transition factor/cluster of differentiation 47, chimeric antigen receptor T, targetiug, cell

中图分类号:

R979.1

王如梦, 斯琴. 靶向c-Met/CD47 CAR-T细胞对卵巢癌细胞的作用[J]. 中国药物警戒, 2024, 21(10): 1103-1112.

WANG Rumeng, SI Qin. Effect of targeting c-Met/CD47 CAR-T cells on ovarian cancer cells[J]. Chinese Journal of Pharmacovigilance, 2024, 21(10): 1103-1112.

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https://zgywjj.magtechjournal.com/CN/Y2024/V21/I10/1103

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