南方医科大学学报

南方医科大学学报 ›› 2023, Vol. 43 ›› Issue (6): 906-914.doi: 10.12122/j.issn.1673-4254.2023.06.05

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低HMGB1含量的肿瘤细胞裂解物增强树突状细胞的抗肺癌作用

潘中武，李思琪，王耀辉，刘海军，桂 琳，董博翰   

  1. 皖南医学院医学微生物学与免疫学教研室，生物化学教研室，药学院，安徽 芜湖 241002
  • 出版日期:2023-06-20 发布日期:2023-07-06

Tumor cell lysate with low content of HMGB1 enhances immune response of dendritic cells against lung cancer in mice

PAN Zhongwu, LI Siqi, WANG Yaohui, LIU Haijun, GUI Lin, DONG Bohan   

  1. Department of Medical Microbiology and Immunology, Department of Biochemistry, School of Pharmacy, Wannan Medical College, Wuhu 241002, China
  • Online:2023-06-20 Published:2023-07-06

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摘要： 目的 探讨低含量高迁移率族蛋白B1（HMGB1）的肿瘤细胞裂解物（TCL）对树突状细胞抗肺癌作用的影响。方法 反复冻融的方法制备Lewis肺癌细胞TCL。Western blot检测TCL中HMGB1蛋白的表达情况。用30 nmol/L甘草酸（GA）抑制Lewis肺癌细胞中HMGB1的表达，进而制备低含量HMGB1的TCL（LH-TCL）。用未处理的Lewis肺癌细胞制备正常含量HMGB1的TCL（NH-TCL）。分为不同剂量NH-TCL负载DC组、LH-TCL负载DC组及PBS对照组，用流式细胞术检测体外诱导的小鼠树突状细胞（DC）表面CD11b、CD11c、CD86表达或细胞凋亡，用酶联免疫吸附试验检测DC细胞IL-12分泌情况。分为NH-TCL刺激DC活化的脾细胞组、LH-TCL刺激DC活化的脾细胞组及PBS对照组，用流式细胞术检测小鼠脾细胞表面CD69的表达情况，酶联免疫吸附试验检测小鼠脾细胞TNF-β的分泌。分为NH-TCL刺激DC活化的脾细胞组、LH-TCL刺激DC活化的脾细胞组及PBS对照组，并设5∶1、10∶1、20∶1三个效靶比进行体外杀伤实验，采用CCK8法检测小鼠脾细胞杀伤肿瘤细胞的活性。分为未活化DC组、NH-TCL活化的DC组、LH-TCL活化的DC组及PBS对照组，通过体内抑瘤实验，评估DC过继免疫细胞疗法抑制小鼠皮下移植瘤生长的效果。结果 GA作用后的Lewis肺癌细胞制备的TCL中，HMGB1的含量显著降低（P<0.05）。与NH-TCL相比，LH-TCL具有更好的减少DC 凋亡的能力（P<0.001），并促进DC表面CD86的表达和IL-12的分泌，而负载LH-TCL的DC也能更好地激活脾淋巴细胞并诱导更强的抗肿瘤免疫作用（P<0.01）。在体外，负载LH-TCL的DC激活脾淋巴细胞对Lewis肺癌细胞的杀伤活性显著提高（P<0.01）；在实验动物体内，免疫负载LH-TCL的DC可明显抑制肿瘤组织生长（P<0.05）。结论 用LH-TCL负载DC，可以增强DC诱导的抗肿瘤免疫作用，提高DC过继免疫细胞治疗的疗效，为该免疫治疗方法的改进提供了一个新的途径。

关键词: 肿瘤细胞裂解物；小鼠脾细胞；肺癌细胞；抗肿瘤免疫；高迁移率族蛋白B1

Abstract: Objective To assess the effect of tumor cell lysate (TCL) with low high-mobility group B1 (HMGB1) content for enhancing immune responses of dendritic cells (DCs) against lung cancer. Methods TCLs with low HMGB1 content (LH-TCL) and normal HMGB1 content (NH-TCL) were prepared using Lewis lung cancer (LLC) cells in which HMGB1 was inhibited with 30 nmol/L glycyrrhizic acid (GA) and using LLC cells without GA treatment, respectively. Cultured mouse DCs were exposed to different doses of NH-TCL and LH-TCL, using PBS as the control. Flow cytometry was used to detect the expressions of CD11b, CD11c and CD86 and apoptosis of the stimulated DCs, and IL-12 levels in the cell cultures were detected by ELISA. Mouse spleen cells were co-cultured with the stimulated DCs, and the activation of the spleen cells was assessed by detecting CD69 expression using flow cytometry; TNF-β production in the spleen cells was detected with ELISA. The spleen cells were then co-cultured with LLC cells at the effector: target ratios of 5:1, 10:1 and 20:1 to observe the tumor cell killing. In the animal experiment, C57/BL6 mouse models bearing subcutaneous LLC xenograft received multiple injections with the stimulated DCs, and the tumor growth was observed. Results The content of HMGB1 in the TCL prepared using GA-treated LLC cells was significantly reduced (P<0.01). Compared with NH-TCL, LH-TCL showed a stronger ability to reduce apoptosis (P<0.001) and promote activation and IL- 12 production in the DCs. Compared with those with NH-TCL stimulation, the DCs stimulated with LH-TCL more effectively induced activation of splenic lymphocytes and enhanced their anti- tumor immunity (P<0.05). In the cell co-cultures, the spleen lymphocytes activated by LH-TCL-stimulated DCs showed significantly enhanced LLC cell killing activity (P<0.01). In the tumor-bearing mice, injections of LH-TCL-stimulated DCs effectively activated host anti-tumor immunity and inhibited the growth of the tumor xenografts (P<0.05). Conclusion Stimulation of the DCs with LH-TCL enhances the anti-tumor immune activity of the DCs and improve the efficacy of DC-based immunotherapy for LLC in mice.

Key words: tumor cell lysates; mouse splenocytes; lung cancer cells; anti-tumor immunity; HMGB1