T细胞激活抑制物免疫球蛋白可变区结构域和程序性死亡受体1分子联合阻断在小鼠结肠癌模型中抗肿瘤效应的研究

Expression and inhibitory effect of T cell activation inhibitor immunoglobulin variable region domain molecules in colorectal carcinomas with K - mosargioma virus gene mutation

目的 探讨T细胞激活抑制物免疫球蛋白可变区结构域分子（VISTA）和CD8、CD33分子在K-鼠类肉瘤病毒癌基因（KRAS）基因外显子不同变异的结直肠癌组织中的表达及VISTA和程序性死亡受体1（PD1）拮抗剂CA170治疗对小鼠CT26结肠癌移植瘤模型抑制作用的机制.方法 收集经病理诊断与KRAS基因测序的结肠癌患者193例,免疫组织化学方法检测上述分子在肿瘤组织中的表达.用小鼠肠癌细胞株CT26构建BALB/c小鼠移植瘤模型,采用液相蛋白分析和流式细胞术和游标卡尺检测两组小鼠模型的多个细胞因子表达和移植瘤体中免疫细胞的变化以及瘤体抑制的指标.统计学分析以上指标.结果 大肠癌组织中VISTA、CD33、CD8分子表达在KRAS基因突变组和野生组之间差异有统计学意义（P =0.020、0.001、0.033）.CA170及对照组瘤体积分别为（1 173.3 ±531.2）、（6 399.4±1 790.5）mm3;两组间瘤体抑制率比较差异有统计学意义（P=0.000）;实验组间的细胞因子粒细胞-巨噬细胞集落刺激因子（GM-CSF）、血管内皮生长因子（VEGF）、MCSF、趋化因子2（CCL2）、干扰素（IFN）-γ、白细胞介素（IL）-10、IL-12以及调节性T细胞（Tregs）、骨髓来源的抑制性细胞（MDSCs）和肿瘤浸润淋巴细胞（TIL）的浸润差异有统计学意义（P =0.010、0.001、0.000）.结论 VISTA和CD33分子高表达及CD8＋的T细胞的低浸润状况与大肠癌的KRAS基因分型相关;VISTA和PD1分子联合阻断剂CA170治疗可以改善肠癌小鼠的全身免疫状况并显著减慢小鼠肠癌的生长.

Objective To investigate the role of T cell activation inhibitor immunoglobulin variable region domain （VISTA） and CD8 and CD33 molecules in the colorectal cancer tissues with different mutations in K-murine sarcoma virus oncogene （KRAS） gene exons expression and mechanisms of inhibition of VISTA and programmed cell death protein （PD1） antagonists CA170 treatment on mouse CT26 colon cancer models.Methods A total of 193 cases of colorectal cancers with pathological diagnosis and KRAS gene sequencing were collected.Immunohistochemistry was used to detect the expression of the above molecules in tumor tissues.The BALB/c mouse tumor model was constructed using the mouse colorectal cancer cell line CT26.The expression of multiple cytokines in the two groups of mouse models and changes of immune cells in the tumor and tumor volume were detected by liquid-phase protein analysis,flow cytometry and vernier caliper,respectively.The results were statistically analyzed.Results There was significant difference in the expression of VISTA,CD33 and CD8 in colorectal cancer tissues between the KRAS mutation group and the wild group （P values of 0.020,0.001,and 0.033 respectively）.Thetumor volume in CA170 and control groups was （1 173.3 ± 531.2）,（6 399.4 ± 1 790.5） mm3.The difference in tumor inhibition rate between the two groups was statistically significant （P =0.000）.There were statistically significant differences in the infiltration of regulatory cells （Tregs）,myeloid-derived suppressor cells （MDSCs）,tumor-infiltrating lymphocytes （TIL） and the expression of cytokines granulocyte macrophage-colony stimulating factor （GM-CSF）,vascular endothelial growth factor （VEGF）,MCSF,chemokine 2 （CCL2）,interferon （IFN）-γ,interleukin （IL）-10,IL-12 between experimental groups （P =0.010,0.001,0.000）.Conclusion The high expression of VISTA and CD33 molecules and the low infiltration of CD8 ＋ T cells correlate with KRAS genotyping of colorectal cancer.VISTA and PD1 antagonists CA170 treatment can improve the systemic immune status of colon cancer mice and significantly slow down the growth of colorectal cancer.