小鼠THBS1基因敲除改善内脏脂肪免疫微环境并抑制结直肠癌腹腔种植转移

Knockout of THBS1 gene improves immune microenvironment of visceral fat and inhibits intraperitoneal implantation and metastasis of colorectal cancer in mice

目的探究血小板反应蛋白1(thrombospondin-1,THBS1)基因敲除对结直肠癌免疫微环境与肿瘤进展的作用。方法构建THBS1基因敲除小鼠,经PCR、琼脂糖凝胶电泳明确小鼠基因型。用THBS1基因野生型(THBS1^(+/+))与THBS1基因敲除(THBS1^(-/-))小鼠构建结直肠癌皮下荷瘤与腹腔种植转移模型,观察THBS1基因敲除对小鼠肿瘤生长情况的影响。用流式细胞术检测腹腔种植转移模型中THBS1基因敲除后对结直肠癌附睾脂肪中免疫细胞的变化。结果与THBS1^(+/+)小鼠相比,THBS1^(-/-)小鼠在结肠癌皮下荷瘤模型中肿瘤生长没有差异(P>0.05),而在腹腔种植转移模型中则显著抑制肿瘤生长(P<0.05)。流式细胞术检测结果显示,与THBS1^(+/+)小鼠相比,THBS1基因敲除后腹腔种植转移模型小鼠的附睾脂肪组织中总巨噬细胞比例增高(P<0.01),其中M1型(经典型或促炎型)巨噬细胞比例升高(P<0.01)、M2型(替代活化型或抗炎型)巨噬细胞比例下降(P<0.01);髓源性抑制细胞(myeloid-derived suppressor cells,MDSC)比例下降(P<0.05),其中Ly6C亚群(P<0.01)比例升高,Ly6G亚群比例降低(P<0.05);CD8+T细胞比例显著升高(P<0.01)、CD4^(+)T细胞比例降低(P<0.01)。与THBS1^(+/+)小鼠相比,THBS1基因敲除小鼠外周血总巨噬细胞、M1型巨噬细胞、M2型巨噬细胞比例均无变化(P>0.05);MDSC及ly6C亚群比例无变化(P>0.05),ly6G亚群比例降低(P<0.05);CD8^(+)T细胞比例显著降低(P<0.05)、CD4+T细胞比例无变化(P>0.05)。结论THBS1基因敲除通过增强肿瘤免疫抑制结直肠癌细胞腹腔种植转移。

Objective To investigate the effects of thrombospondin1(THBS1)gene knockout on immune microenvironment and tumor progression of colorectal cancer.Methods THBS1 gene knockout(THBS1^(-/-))mice were constructed and its genotypes were determined by PCR and agarose gel electrophoresis.Subcutaneous tumor bearing and intraperitoneal implantation metastasis models of colorectal cancer were constructed in wild type(WT)and THBS1^(-/-)mice to observe the effect of THBS1 on tumor growth.Immune cells in epididymal adipose of tumor bearing mice of both types were detected by flow cytometry.Results Compared with the WT mice,THBS1^(-/-)mice showed no difference in tumor growth in colorectal cancer subcutaneous tumor bearing model(P>0.05),but significantly inhibited tumor growth in intraperitoneal implantation metastasis model(P<0.05).Subsequently,flow cytometry indicated that the proportion of total macrophages in the epididymal adipose tissue was increased in the THBS1^(-/-)mice than the WT mice(P<0.01),with the proportion of M1-like macrophages(typical or proinflammatory type)increased(P<0.01)and that of M2-like macrophages(alternately activated or anti-inflammatory type)decreased(P<0.01).The proportion of myeloid-derived suppressor cells(MDSC)was decreased(P<0.05),that of Ly6C subgroup was increased(P<0.01),that of Ly6G subgroup was decreased(P<0.05),that of CD8^(+)T cells was significantly increased(P<0.01),and that of CD4^(+)T cells was decreased(P<0.01).There were no changes in the proportions of total macrophages,M1-like macrophages and M2-like macrophages in the peripheral blood of intraperitoneal implantation metastasis model mice(P>0.05).The proportions of MDSC and ly6C subgroup were not changed(P>0.05),while that of ly6G subgroup were decreased(P<0.05).The proportion of CD8^(+)T cells was significantly decreased(P<0.05),while that of CD4^(+)T cells was not changed(P>0.05).Conclusion THBS1 gene knockout inhibits the intraperitoneal metastasis of colorectal cancer cells by enhancing tumor immunity.