低剂量辐射对Lewis肺癌小鼠肿瘤微环境的影响

Effects of low-dose radiation on tumor microenvironment in mice bearing Lewis lung carcinoma tumors

目的探讨低剂量辐射(LDR)对Lewis肺癌小鼠肿瘤微环境(TME)的影响,以及高剂量辐射(HDR)前LDR预处理在TME中发挥的效应机制。方法接种Lewis肺癌细胞(1×10^(6))于雄性C57BL/6小鼠左侧腋下,待小鼠肿瘤直径1cm左右(12d),随机分为假手术Sham(1)组、LDR(2)组、HDR(3)组和HDR前LDR预处理组LDR-HDR(4)组。接种Lewis肺癌细胞后第14天和第17天同时间点各组分别处死5只小鼠,剥离肿瘤组织并摘取脾脏,检测脾脏及肿瘤组织中骨髓源抑制细胞(MDSC)、肿瘤浸润淋巴细胞(TIL)、肿瘤组织中转化生长因子β(TGF-β)和基质细胞衍生因子1(SDF-1)含量;取小鼠眼球血,检测血清TGF-β含量。采用Shapiro-Wilk法进行正态分布检验,组间两两比较采用LSD-t检验。结果各组小鼠脾脏MDSC第14天差异有统计学意义,F=29.666,P<0.001;组间比较P_(1-2)=0.003、P_(1-3)<0.001、P_(3-4)=0.030。17d4组间均值差异有统计学意义,F=19.203,P<0.001;组间比较P_(1-2)=0.029、P_(1-3)<0.001、P_(3-4)=0.035。肿瘤MDSC 14d4组差异有统计学意义,F=13.800,P<0.001;组间比较P_(1-2)=0.011、P_(1-3)=0.003、P_(3-4)=0.031。17d4组均值差异有统计学意义,F=17.508,P<0.001;组间两两比较P_(1-2)=0.002、P_(1-3)=0.004、P_(3-4)=0.033。第14天各组CD4+/CD8+T细胞数值组间均值差异有统计学意义,F=19.110,P<0.001;组间比较P_(1-2)<0.001、P_(1-3)=0.016、P_(3-4)=0.001。17d4组均值差异有统计学意义,F=45.644,P<0.001;组间两两比较P_(1-2)<0.001、P_(1-3)<0.001、P_(3-4)=0.027。14d各组肿瘤中SDF-1均值差异有统计学意义,F=29.099,P<0.001;组间两两多重比较P_(1-2)=0.005、P_(1-3)<0.001、P_(3-4)=0.804。17d4组均值差异有统计学意义,F=22.147,P<0.001;组间比较P_(1-2)=0.004、P_(1-3)=0.001、P_(3-4)=0.231。14d各组血清TGF-β组间均值差异有统计学意义,F=30.243,P<0.001;组间两两多重比较P_(1-2)<0.001、P_(1-3)<0.001、P_(3-4)<0.001。17d4组均值差异有统计学意义,F=39.030,P<0.001;组间比较P_(1-2)<0.001、P_(1-3)=0.022、P_(3-4)=0.023。14d各组肿瘤TGF-β均值差异有统计学意义,F=13.773,P<0.001;两两多重比较P_(1-2)=0.008、P_(1-3)=0.007、P_(3-4)<0.001。17d4组均值差异有统计学意义,F=5.595,P=0.008;组间两两比较P_(1-2)=0.012、P_(1-3)=0.369、P_(3-4)=0.024。结论LDR可增加Lewis肺癌小鼠TME中的抗肿瘤免疫效应;LDR的免疫适应性具有逆转TME中的免疫抑制的作用。

Objective To investigate the effect of low-dose radiation(LDR)on tumor microenvironment(TME)in murine Lewis lung carcinoma model and the mechanism initiated by LDR pretreatment before high-dose radiation(HDR)in TME.Methods Lewis lung carninoma(LLC)cells(1×10^(6))were inoculated into the left flank of the male mice,until the tumors about 1 cm in diameter(12 d),the mice were randomly divided into Sham(1)operation group,low-dose radiation group(LDR)(2),high-dose radiation group(HDR)(3)and low-dose radiation pretreatment group before high-dose radiation group(LDR-HDR)(4)10 patients/group.Five mice in each group were sacrificed at the same time point on day 14 and 17 after inoculation of Lewis lung cancer cells,stripping the tumor tissues and spleens and detecting the proportion of myeloid-derived suppressor cells MDSC.Testing the tumor infiltrating lymphocyte(TIL),the contents of transforming growth factor-β(TGF-β)and stromal cell-derived factor-1(SDF-1)of Tumor tissues;Blood was collected from ophthalmic vein.Normal distribution test was performed by Shapiro-Wilk,and LSD-t test was used for pair comparison between groups.Results The mean comparison between groups was statistically significant(F=29.666,P<0.001),and the comparison between groups was P_(1-2)=0.003,P_(1-3)<0.001,P_(3-4)=0.030.The data test results of the four groups on day17 were consistent with normal distribution,and the mean difference between groups was statistically significant(F=19.203,P<0.001),and the comparison between groups was P_(1-2)=0.029,P_(1-3)<0.001,P_(3-4)=0.035.the mean comparison between groups was statistically significant(F=13.800,P<0.001),and the comparison between groups was P_(1-2)=0.011,P_(1-3)=0.003,P_(3-4)=0.031.The data test results of the four groups on day 17 were consistent with normal distribution,and the mean difference between groups was statistically significant(F=17.508,P<0.001),and the comparison between groups was P_(1-2)=0.002,P_(1-3)=0.004,P_(3-4)=0.033.The mean comparison between groups was statistically significant(F=29.099,P<0.001),and the comparison between groups was P_(1-2)=0.005,P_(1-3)<0.001,P_(3-4)=0.804.The data test results of the four groups on day 17 were consistent with normal distribution,and the mean difference between groups was statistically significant(F=22.147,P<0.001),and the comparison between groups was P_(1-2)=0.004,P_(1-3)=0.001,P_(3-4)=0.231.The mean comparison between groups was statistically significant(F=30.243,P<0.001),and the comparison between groups was P_(1-2)<0.001,P_(1-3)<0.001,P_(3-4)<0.001.The data test results of the four groups on day 17 were consistent with normal distribution,and the mean difference between groups was statistically significant(F=39.030,P<0.001),and the comparison between groups was P_(1-2)<0.001,P_(1-3)=0.022,P_(3-4)=0.023.The mean comparison between groups was statistically significant(F=13.773,P<0.001),and the comparison between groups was P_(1-2)=0.008,P_(1-3)=0.007,P_(3-4)<0.001.The data test results of the four groups on day 17 were consistent with normal distribution,and the mean difference between groups was statistically significant(F=5.595,P=0.008),and the comparison between groups was P_(1-2)=0.012,P_(1-3)=0.369,P_(3-4)=0.024.Conclusions LDR increases the anti-tumor immune effect of TME in Lewis lung cancer mice.The immune adaptation of LDR can reverse the immunosuppression in TME.