脂多糖通过调节浸润性淋巴细胞抑制小鼠肺腺癌的进展

Lipopolysaccharide suppresses the progression of lung adenocarcinoma by regulating infiltrating lymphocytes in mice

目的:观察脂多糖(LPS)对小鼠肺腺癌进展的影响,并探讨相关免疫学机制。方法:小鼠Lewis肺癌(LLC)细胞分为3组,分别给予PBS或LPS(10、20 mg/L)处理0、24、48 h,采用CCK-8法检测细胞增殖,采用Annexin-V/PI双染法检测处理48 h后细胞凋亡率。取10只6~8周龄C57BL/6J雌性小鼠,皮下注射LLC细胞构建移植瘤小鼠模型,成瘤后将小鼠均分为两组,每组5只;分别腹腔注射PBS(对照组)或LPS(LPS组),每3 d注射1次,共注射4次。小鼠第1次腹腔注射PBS或LPS 6 h后,尾静脉取血,采用ELISA法检测小鼠血清白细胞介素-6(IL-6)、IL-1β、肿瘤坏死因子-α(TNF-α)的表达。末次注射后第3天处死小鼠,测量肿瘤体积及质量。采用荧光抗体标记法检测小鼠肿瘤组织中CD45^(+)淋巴细胞、CD3^(+)T淋巴细胞、CD3^(+)CD8^(+)T淋巴细胞比例及CD8^(+)T淋巴细胞表面细胞程序性死亡蛋白1(PD-1)、T淋巴细胞免疫球蛋白黏蛋白分子3(TIM-3)的表达水平。结果:LPS体外对LLC细胞的增殖、凋亡无影响(P>0.05)。与对照组比较,LPS组小鼠肿瘤体积和质量均减小,血清IL-6、IL-1β和TNF-α水平升高(P均<0.05);LPS组小鼠肿瘤组织中CD3^(+)CD8^(+)T淋巴细胞比例增加,而CD8^(+)T淋巴细胞表面PD-1、TIM-3表达降低(P均<0.05)。结论:LPS可能通过促进浸润性淋巴细胞增多并降低CD8^(+)T淋巴细胞表面PD-1、TIM-3的表达,抑制小鼠体内肺腺癌移植瘤的生长。

Aim:To observe the effects of lipopolysaccharide(LPS)on mouse lung adenocarcinoma progression,and to explore the relevant immunological mechanisms.Methods:Lewis lung cancer(LLC)cells were treated with phosphate buffer(PBS)or LPS(10,20 mg/L)for 0,24,48 hours.The cell proliferation was examined by CCK-8 assay,and apoptosis of LLC cells at the end of 48 hour treatment was examined by Annexin V/PI double staining.Ten female C57BL/6J mice with age ranging 6-8 weeks were selected and injected with LLC cells.Once tumors were detected,mice were allocated into 2 groups with an intraperitoneal injection of PBS or LPS once every 3 days for a total of 4 injections.The peripheral blood samples were collected at the 6th hour after the 1st injection and the levels of interleukin-6(IL-6),IL-1β,and tumor necrosis factor-α(TNF-α)in serum were measured by ELISA.Mice were killed at the 3rd day after the last injection to measure the volume and mass of tumors;the proportion of infiltrating CD45^(+)lymphocytes,CD3^(+)T lymphocytes,CD3^(+)CD8^(+)T lymphocytes and the levels of programmed cell death protein 1(PD-1)and T lymphocyte immunoglobulin and mucin domain-containing protein 3(TIM-3)on CD8^(+)T lymphocytes were measured by fluorescent antibody labeling.Results:LPS had no effects on the proliferation or apoptosis of LLC cells(P<0.05).Compared with control group,the levels of IL-6,IL-1β,and TNF-αin the serum of mice were significantly increased,and the tumor volume and mass in LPS group decreased significantly(P<0.05).Compared with control group,the proportion of CD3^(+)CD8^(+)T lymphocytes increased significantly in LPS group,while the expressions of PD-1 and TIM-3 on CD8^(+)T lymphocytes were significantly reduced(P<0.05).Conclusion:LPS might inhibit the growth of mouse lung adenocarcinoma by increasing the proportion of infiltrating lymphocytes in tumor tissue and reducing the expressions of PD-1 and TIM-3 on CD8^(+)T lymphocytes.