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

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

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。

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-βproduction 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 DCbased immunotherapy for LLC in mice.