多西他赛联合胸腺肽α1对大鼠乳腺癌免疫微环境中Treg数量的影响及其机制研究

Effects of docetaxel combined with thymosinα1 on number of Treg in immune microenvironment of rats with breast cancer and its mechanism

背景与目的:乳腺癌患者体内存在严重的免疫失衡,胸腺肽α1(thymosinα1,Tα1)作为免疫增强剂发挥作用,探究多西他赛(docetaxel,DCT)联合Tα1对大鼠乳腺癌免疫微环境中调节性T细胞(regulatory T cell,Treg)数量的影响,并初步分析其作用机制。方法:随机将60只雌性SD大鼠分为模型组、DCT组(剂量为10 mg/kg)、Tα1组(剂量为0.8 mg/kg)及3个DCT+Tα1组(DCT剂量为10 mg/kg,Tα1剂量分别为0.2、0.4、0.8 mg/kg),每组10只。给雌性SD大鼠接种大鼠乳腺癌细胞系SHZ-88建立移植瘤模型,成瘤后,DCT组、Tα1组及DCT+Tα1组分别腹腔注射相应剂量的DCT、Tα1,给药周期均为20 d,1次/d。测量大鼠肿瘤体积,采用原位末端转移酶标记(TdT-mediated dUTP nick end labeling,TUNEL)染色法检测肿瘤细胞的凋亡情况。采用流式细胞术检测大鼠肿瘤组织中CD4^(+)CD25^(+)Foxp3^(+)Treg数量及表达程序性死亡[蛋白]-1(programmed death-1,PD-1)的Treg数量,采用酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测肿瘤组织中白细胞介素-10(interleukin-10,IL-10)、转化生长因子-β(transforming growth factor-β,TGF-β)的表达,采用蛋白质印迹法(Western blot)检测肿瘤组织中PD-1及程序性死亡[蛋白]配体-1(programmed death ligand-1,PD-L1)的表达。结果:与模型组相比,给药组均可明显抑制肿瘤的生长,促进肿瘤细胞凋亡,显著下调CD4^(+)CD25^(+)Foxp3^(+)Treg数量,以及IL-10、TGF-β、PD-1和PD-L1的表达(P<0.05),其中DCT+0.8 mg/kg Tα1组作用效果最显著,明显优于其他给药组。结论:DCT联合Tα1可抑制大鼠肿瘤的生长,其中以DCT+0.8 mg/kg Tα1效果最显著,作用机制可能是通过下调PD-1/PD-L1的表达,抑制CD4^(+)CD25^(+)Foxp3^(+)Treg浸润。

Background and purpose:There is severe immune imbalance in breast cancer patients.Thymosinα1(Tα1)acts as an immune enhancer.The aim of this study was to explore the effects of docetaxel(DCT)combined with Tα1 on number of regulatory T cell(Treg)in immune microenvironment of rats with breast cancer,and preliminarily analyze its action mechanism.Methods:Sixty female SD rats were randomly divided into model group,DCT group(10 mg/kg),Tα1 group(0.8 mg/kg)and three DCT+Tα1(10 mg/kg DCT;0.2,0.4,0.8 mg/kg Tα1)groups with 10 cases in each group.SD female rats were inoculated with breast cancer cell line SHZ-88 to construct xenograft models.After tumor formation,DCT group,Tα1 group and DCT+Tα1 group were given intraperitoneal injection of DCT and Tα1 respectively.The administration period was 20 d,once a day.The tumor volume was measured.TdT-mediated dUTP nick end labeling(TUNEL)staining was applied to detect apoptosis of tumor cells.The number of CD4^(+)CD25^(+)Foxp3^(+)Treg,expression of programmed death-1(PD-1)and number of Treg in tumor tissues were detected by flow cytometry.The expressions of interleukin-10(IL-10)and transforming growth factor-β(TGF-β)in tumor tissues were detected by enzyme-linked immunosorbent assay(ELISA).The expressions of PD-1 and programmed death ligand-1(PD-L1)in tumor tissues were detected by Western blot.Results:Compared with model group,drug administration effectively inhibited tumor growth,promoted apoptosis of tumor cells and significantly down-regulated the number of CD4^(+)CD25^(+)Foxp3^(+)Treg and expressions of IL-10,TGF-β,PD-1 and PD-L1(P<0.05).The action effect in DCT+0.8 mg/kg Tα1 group was the most significant,which was significantly better compared with the other administration groups.Conclusion:DCT combined with Tα1 can inhibit growth of rat tumor.The effect of DCT+0.8 mg/kg Tα1 is the most significant,and its mechanism may involve down-regulating expression of PD-1/PD-L1 and inhibiting infiltration of CD4^(+)CD25^(+)Foxp3^(+)Treg.