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中国癌症防治杂志

中国癌症防治杂志 ›› 2023, Vol. 15 ›› Issue (5): 532-537.doi: 10.3969/j.issn.1674-5671.2023.05.10

• 基础研究 • 上一篇    下一篇

Galectin-4调控PD-L1影响CD8+ T细胞杀伤肝癌细胞的功能

  

  1. 徐州市中心医院肿瘤科；徐州医科大学第二附属医院疼痛科；徐州市中心医院呼吸科
  • 出版日期:2023-10-25 发布日期:2023-11-03
  • 通讯作者: 刘媛媛 E-mail：hbulyy@126.com
  • 基金资助:
    江苏省研究生科研创新计划项目（KYCX20?2494）

 Galectin-4 regulates PD-L1 and affects the function of CD8+T cells in killing liver cancer cells

  • Online:2023-10-25 Published:2023-11-03

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摘要： 目的 探讨人半乳糖凝集素4（Galectin⁃4，Gal⁃4）影响CD8+T细胞杀伤肝癌细胞的功能及机制。方法 提取并鉴定人外周血CD8+T细胞及树突状细胞（dendritic cells，DC）。选取人正常肝细胞WRL68和肝癌细胞HepG2、SMMC⁃7721、Hep3B、Huh⁃7，并利用Western blot法检测Gal⁃4蛋白的表达。利用Lipofectamine 3000试剂在肝癌细胞HepG2和SMMC⁃7721中转染Gal⁃4 siRNA及其阴性对照（si⁃Gal⁃4组和si⁃NC组），si⁃Gal⁃4组和si⁃NC组HepG2和SMMC⁃7721细胞分别与被DC细胞活化的CD8+T细胞共孵育18 h，依次记为si⁃Gal⁃4/HepG2+CD8+T组、si⁃NC/HepG2+CD8+T组、si⁃Gal⁃4/SMMC⁃7721+CD8+T组、si⁃NC/SMMC⁃7721+CD8+T组。采用细胞毒性实验检测CD8+T细胞对各组肿瘤细胞的杀伤能力，酶联免疫吸附实验（enzyme⁃linked immunosorbent assay，ELISA）检测各组细胞上清液中干扰素（interferon⁃γ，INF⁃γ）和肿瘤坏死因子⁃α（tumor necrosis factor⁃α，TNF⁃α）的浓度，Western blot法检测各组肿瘤细胞中PD⁃L1蛋白的表达。结果 相比于正常肝细胞WRL68，肝癌细胞HepG2、SMMC⁃7721、Hep3B、Huh⁃7中Gal⁃4蛋白表达水平均显著上调（均P<0.001）；相比于si⁃NC/HepG2+CD8+T组或si⁃NC/SMMC⁃7721+CD8+T组，si⁃Gal⁃4/HepG2+CD8+T组和si⁃Gal⁃4/SMMC⁃7721+CD8+T组CD8+T细胞杀伤能力均显著增加（均P<0.001），且抑制Gal⁃4表达后，共培养体系中INF⁃γ和TNF⁃α的浓度均显著增加（均P<0.001）。相比于si⁃NC组，si⁃Gal⁃4组HepG2和SMMC⁃7721细胞PD⁃L1蛋白表达显著下调（P<0.001）。结论 Gal⁃4在肝癌细胞中高表达，抑制Gal⁃4可能下调肝癌细胞PD⁃L1的表达，进而促进CD8+T细胞的杀伤能力。

关键词:  , 肝癌；人半乳糖凝集素4；CD8+T细胞；PD-L1

Abstract: Objective To investigate the effect of human Galectin⁃4 (Gal⁃4) on CD8+T cells killing liver cells and its mechanism. Methods The human peripheral blood CD8+T cells and dendritic cells (DC) were extracted and identified. Normal human liver cells WRL68 and liver cancer cells including HepG2, SMMC⁃7721, Hep3B and Huh⁃7 were selected, and Western blot was used to detect the expression of Gal⁃4 protein. The liver cancer cells HepG2 and SMMC⁃7721 were transfected with Gal⁃4 siRNA and negative control (the si⁃Gal⁃4 group and si⁃NC group) using Lipofectamine 3000. The HepG2 and SMMC⁃7721 cells in the si⁃Gal⁃4 and si⁃NC groups were co⁃incubated with DC⁃activated CD8+T cells for 18 hours (the si⁃Gal⁃4/HepG2+CD8+T group, si⁃NC/HepG2+CD8+T group, si⁃Gal⁃4/SMMC⁃7721+CD8+T group, si⁃NC/SMMC⁃7721+CD8+T group). Cytotoxicity experiments were performed to evaluate the capability of CD8+T cells in killing the tumor cells in each group, and enzyme⁃linked immunosorbent assay (ELISA) was used to detect the levels of interferon ⁃γ (INF⁃ γ) and tumor necrosis factor⁃α (TNF⁃α) in the cell supernatants of each group. Western blot was used to detect the expression of PD⁃L1 protein in the tumor cells of each group. Results Compared with normal liver cell WRL68, the expression of Gal⁃4 protein were significantly up⁃regulated in liver cancer cells HepG2, SMMC⁃7721, Hep3B, and Huh⁃7 (all P<0.001). Compared with the si⁃NC/HepG2+CD8+T group or si⁃NC/SMMC⁃7721+CD8+T group, the killing ability of si⁃Gal⁃4/HepG2+CD8+T group and the si⁃Gal⁃4/SMMC⁃7721+CD8+T group were significant increased (all P<0.001). After inhibiting the expression of Gal⁃4, the concentrations of INF⁃γ and TNF⁃α in co⁃culture system were significantly increased (all P<0.001). Compared with the si⁃NC group, the expression of PD⁃L1 protein in HepG2 and SMMC⁃7721 cells of si⁃Gal⁃4 group were significantly down⁃regulated (P<0.001). Conclusions Gal⁃4 is highly expressed in liver cancer cells, and inhibition of Gal⁃4 may down⁃regulate the expression of PD⁃L1 in liver cancer cells and promote the killing ability of CD8+T cells.

Key words:  Liver cancer, Galectin-4, CD8+T cells, PD-L1

中图分类号: 

  • R735.7