不同范围冷消融治疗对Lewis肺癌小鼠Tregs细胞的影响

Effects of different ranges of cold ablation on Lewis lung cancer mouse Tregs cells

目的探究不同范围冷消融治疗对Lewis肺癌小鼠Tregs细胞的影响。方法采用Lewis小鼠肺腺癌细胞株建立C57BL/6小鼠皮下移植瘤模型,选取造模成功的小鼠随机分为对照组、冷消融低范围组、冷消融中范围组、冷消融全范围组,每组5只。冷消融组予以不同冷冻范围的冷消融手术,冷消融低范围组消融面积为肿瘤的50%~60%,中范围组为60%~90%,全范围组为100%;对照组予以假手术治疗。术后14日取外周血、脾脏及肿瘤组织。流式细胞仪检测肿瘤组织、脾脏中CD4^(+)CD25+FoxP3^(+)T细胞数量;Western blot法检测肿瘤组织中Foxp3蛋白表达情况;qRT-PCR检测肿瘤组织中Foxp3 mRNA表达;ELISA检测外周血中细胞因子白介素(IL)-2、转化生长因子(TGF)-β的表达。结果与对照组相比,冷消融中范围组脾脏组织中CD4^(+)CD25+FoxP3^(+)T细胞比例降低(P<0.05),冷消融各组组间相比差异无统计学意义(P>0.05);与对照组相比,冷消融全范围组肿瘤组织中CD4^(+)CD25+FoxP3^(+)T细胞比例降低(P<0.05),冷消融各组组间相比差异无统计学意义(P>0.05)。与对照组相比,冷消融各组Foxp3蛋白、mRNA表达低(P<0.05),中范围、全范围组低于低范围组(P<0.05),中范围组与全范围组相比差异无统计学意义(P>0.05)。与对照组相比,冷消融各组外周血中IL-2水平均高(P<0.05),中范围组、全范围组均高于低范围组(P<0.05),全范围组低于中范围组(P<0.05)。与对照组相比,冷消融低范围组、中范围组、全范围组TGF⁃β水平均低(P<0.05),低范围组、全范围组均高于中范围组(P<0.05),全范围组高于低范围组和中范围组(P<0.05)。结论冷消融治疗不仅可直接引起肿瘤组织机械性损伤,而且可能通过抑制Tregs细胞表达抑制残余肿瘤的生长;冷消融范围60%~100%的效果优于冷消融低范围50%~60%。

Objective To investigate the effects of cold ablation of different ranges on the Tregs cells of Lewis lung cancer mice.Methods The subcutaneous transplantation tumor model of C57BL/6 mice was developed using Lewis mouse lung adenocarcinoma cell lines.Five mice were randomly assigned to four groups,including control group,low-range cryoablation group,medium-range cryoablation group,and full-range cryoablation group.The cold ablation group was given cold ablation with different freezing ranges,the ablation area of the low-range group was 50%~60%of the tumor,the middle-range group was 60%~90%,and the full-range group was 100%,whereas the control group underwent sham surgery.Periperalblood,spleen,and tumor tissue were collected 14 days after surgery.The number of CD4^(+)CD25+FoxP3^(+)T cells in tumor tissue and spleen was determined using flow cytometry.Western blot was used to detect the expression of Foxp3 protein in tumor tissues,qRT-PCR to detect the expresssion of Foxp3 mRNA in tumor tissues.In addition,the expression of cytokines IL-2 and TGF-βin peripheral blood was detected using ELISA.Results Compared with the control group,the proportion of CD4^(+)CD25+FoxP3^(+)T cells in the spleen tissue of the middle-range of cold ablation group was decreased with difference of three cold ablation groups statistically significant(P<0.05).Compared with the control group,the proportion of CD4^(+)CD25+FoxP3^(+)T cells in the whole range of cold ablation group was decreased with the difference of three cold ablation groups not statistically significant(P>0.05).Compared with the control group,the expressions of Foxp3 protein and mRNA in cold ablation groups were lower(P<0.05),and those in the middle and full range groups were lower than those in the low range group(P<0.05).There was no statistical difference between the middle range group and the full range group(P>0.05).Compared with the control group,the level of IL-2 in peripheral blood of each cold ablation group was higher(P<0.05),the middle range group and the full range group were higher than the low range group(P<0.05),and the full range group was lower than the middle range group(P<0.05).Compared with the control group,the level of TGF-βin the low range group,the middle range group and the full range group was lower(P<0.05).The low range group and the full range group were higher than the middle range group(P<0.05),and the full range group was higher than the low range group and the middle range group(P<0.05).Conclusion In addition to directly causing mechanical damage to tumor tissue,cold ablation can also inhibit the growth of the residual tumor by inhibiting the expression of Tregs cells.The efficacy of cold ablation groups in the range of 60%~100%was superior to that of low range.The effect of cold ablation in the range of 60%~100%is better than that in the low range of 50%~60%.