敲低卷曲跨膜受体蛋白2抑制小鼠舌鳞癌并增强CTLA4单抗疗效的研究

Knockdown of Frizzled 2 inhibits mouse tongue squamous carcinoma and enhances CTLA4 monoclonal antibody efficacy

目的探讨卷曲跨膜受体蛋白2(FZD2)在小鼠舌鳞癌中的功能,及其对细胞毒性T淋巴细胞相关抗原4(CTLA4)单抗治疗的影响。方法细胞实验:SCCVII小鼠舌鳞癌细胞分为空白对照组(转染LV-kdCon)和实验组(转染LV-kdFZD2)。用细胞计数试剂盒-8(CCK-8)检测细胞增殖能力,用Transwell小室实验检测迁移能力,用人脐静脉内皮细胞验证血管生成能力。动物实验:雌性C3H/He小鼠构建荷瘤小鼠模型,分为对照组[给予磷酸盐缓冲液注射(PBS)]、kdFZD组(接种实验组细胞并给予等量PBS注射)、αCTLA4组(小鼠皮下接种空白对照组细胞并给予CTLA4单抗注射)和kdFZD+αCTLA组(小鼠皮下接种实验组细胞并给予CTLA4单抗注射)。用定量聚合酶链反应检测肿瘤组织mRNA相对表达水平,测量肿瘤体积和质量。结果空白对照组和实验组细胞中FZD2 mRNA相对表达水平分别为1.01±0.18和0.52±0.18,细胞迁移数为(466.70±44.10)和(160.00±26.46)个,人脐静脉内皮上皮细胞形成的小管分支节点数分别为(108.70±6.35)和(84.33±10.02)个,总毛细血管长度分别为(20235±2229)和(16549±338)μm,组间比较,在统计学上差异均有统计学意义(P<0.05,P<0.01)。对照组、kdFZD组、αCTLA4组和kdFZD+αCTLA组肿瘤体积分别为(449.50±114.80)、(131.10±13.49)、(83.62±26.47)和(2.45±0.91)mm^(3),肿瘤质量分别为(0.78±0.11)、(0.22±0.06)、(0.13±0.02)和(0.03±0.01)g,组间比较,在统计学上差异均有统计学意义(P<0.001,P<0.0001)。结论FZD2可作为潜在的舌鳞癌治疗靶点,有望成为舌鳞癌辅助免疫治疗优化的关键分子。

Objective To investigate the function of Frizzled 2(FZD2)in mouse tongue squamous cell carcinoma and its effect on cytotoxic T lymphocyte-associated antigen 4(CTLA4)monoclonal antibody.Methods Cell experiment:SCCVII mouse tongue squamous cell carcinomq cells were divided into blank control group(transfected LV-kdCon)and experimental group(transfected LV-kdFZD2).Cell proliferation ability was detected by cell counting kit-8(CCK-8);migration ability was detected by Transwell cell assay;and angiogenesis ability was verified by human umbilical vein endothelial cells.Animal experiments:Female C3H/He mice were constructed with tumor bearing mice model.Mice were divided into control group[injected with phosphate buffer solution(PBS)],kd FZD group(inoculated with experimental cells and injected with an equal amount of PBS),αCTLA4 group(inoculated with blank control cells subcutaneously and injected with CTLA4monoclonal antibody)and kd FZD+αCTLA group(inoculated with experimental cells subcutaneously and injected with CTLA4 monoclonal antibody).The mRNA expression of tumor tissue was detected by quantitative polymerase chain reaction(q PCR),and the tumor volume and mass were measured.Results The relative expression levels of FZD2mRNA in the control group and the experimental group were 1.01±0.18 and 0.52±0.18;the cell migration numbers were 466.70±44.10 and 160.00±26.46;the number of branch nodes of human umbilical vein endothelial cells were108.70±6.35 and 84.33±10.02;the total capillary length were(20235±2229)and(16549±338)μm,respectively,with statistical significance(P<0.05,P<0.01).The tumor volumes of control group,kd FZD group,αCTLA4 group and kd FZD+αCTLA group were(449.50±114.80),(131.10±13.49),(83.62±26.47)and(2.45±0.91)mm^(3);the tumor weights were(0.78±0.11),(0.22±0.06),(0.13±0.02)and(0.03±0.01)g,respectively,and the differences were statistically significant(P<0.001,P<0.0001).Conclusion FZD2 can be used as a potential therapeutic target for tongue squamous cell carcinoma and is expected to be a key molecule in the optimization of adjuvant immunotherapy for tongue squamous cell carcinoma.