B、T淋巴细胞弱化因子胞外段联合热休克蛋白70-子宫颈癌TC-1细胞抗原肽复合物治疗小鼠子宫颈癌模型的效果  

Therapeutic effects of B and T lymphocyte attenuator extracellular domain and heat shock protein 70 antigen peptide on cervical cancer in mouse model

作　　者：韩凌斐[1] 邱伟民[1] 胡程[1] 王玲[1] 姚红霞[1] 熊诗诣[1] 孟梦[1] 方勇[2] 马丁[2] 

机构地区：[1]同济大学附属第一妇婴保健院妇科,上海200040 [2]华中科技大学同济医学院附属同济医院妇产科

出　　处：《中华妇产科杂志》2010年第8期603-608,共6页Chinese Journal of Obstetrics and Gynecology

基　　金：国家重点基础研究发展计划（2009CB521800）;同济大学青年优秀人才培养行动计划（2009KJ108）

摘　　要：目的探讨B、T淋巴细胞弱化因子（BTLA）胞外段联合热休克蛋白（HSP）70-宫颈癌TC-1细胞（HSP70-TC-1）抗原肽复合物治疗小鼠宫颈癌模型的效果。方法（1）将TC-1细胞接种于C57BL／6小鼠成瘤后，实时荧光定量PCR技术检测小鼠肿瘤组织中BTLA、疱疹病毒侵入介质（HVEM）mRNA的表达，流式细胞仪检测肿瘤组织中浸润的淋巴细胞细胞膜表面BTLA、HVEM的表达（以荧光强度表示）。（2）小鼠接种TC-1细胞后，根据处理方法的不同分为5组，即pcDNA3．1（注射空载体pcDNA3．1质粒，作为对照）、psBTLA（注射表达BTLA胞外段的真核表达载体psBTLA质粒）、HSP70（注射HSP70-TC-1抗原肽复合物）、HSP70＋pcDNA3．1（注射HSP70-TC-1抗原肽复合物和pcDNA3．1质粒）、HSP70’psBTLA（注射HSP70-TC-1抗原肽复合物和psBTLA质粒）组，观察各组小鼠体内肿瘤的生长情况；并采用实时荧光定量PCR技术检测各组肿瘤组织中相关免疫基因包括．y干扰素（IFN-γ）、白细胞介素（IL）2、IL-10、转化生长因子B（TGF—β）和人叉头型基因P3（FoxP3）mRNA的表达；免疫组化法计数各组瘤旁组织中CD8＋T淋巴细胞；7-氨基放线菌素D／羧基荧光素乙酰乙酸琥珀酰亚胺酯双标法检测各组脾淋巴细胞的杀伤效应（以杀伤率表示）；氚标记胸腺嘧啶核苷掺入法测定各组脾淋巴细胞的增殖活性，酶联免疫吸附试验测定各组脾淋巴细胞培养上清液中IL-2和IFN-γ的浓度。结果（1）小鼠接种TC-1细胞后第7、14、21、28天，其肿瘤组织中BTLAmRNA的表达水平逐渐上调，第14天达最高，为2．83±0．35，与第7天的1．66±0．25比较，差异有统计学意义（P〈0．05）；而HVEMmRNA的表达水平无明显变化（P〉0．05）。接种TC-1细胞后第7、14天，肿瘤组织中浸润的淋巴细胞细胞膜表面BTLA的平均荧光强度分别为33．5和51．8，两者比较，差异有统计学意义（P〈0Objective To investigate the synergistic therapy effects of B and T lymphocyte attenuator（BTLA） extracellular domain in combination with heat shock protein 70 （HSP70）-TC-1 antigen peptide complex on the mouse model of cervical cancer and the related immunological mechanisms. Methods （1）Detecting the BTLA and herpesvirus entry mediator （HVEM） geue expression in the tumor ml after C57BL/6 mice were inoculated with TC-1 tumor cells by realtime PCR; BTLA, HVEM expression on tumor infiltrating lymphocytes cell surface were detected by flow cytometry （fluorescence intensity）. （2）According to different treatments, tumor-bearing mice were divided into 5 groups, which was injected with pcDNA3.1 （ empty vector plasmid as control ）, psBTLA （ vector plasmid which expresses BTLA extracellular domain ）, HSP70 （HSP70-TC-1 cell peptide complex ）, HSPT0 ＋ pcDNA3.1 or HSP70 ＋ psBTLA, respectively. The weight of tumor was recorded. The expression of immunoregulatory genes in tumor microenvironment were detected. The change of lymphocyte amount and cytotoxicity were detected too; lymphocyte .proliferation activity was measured by tritium thymidine incorporation assay; the concentration of interleukin （IL）2 and interferon-γ （IFN-γ） in supernatants of spleen lymphocyte were measured by enzyme-linked immunosorbent assay （ ELISA ）. Results （ 1 ） BTLA gene expression was gradually increased after tumor cells inoculation. The highest expression level was 2. 83 ±0. 35 at 14th day, which had statistical significance difference with the 7th day expression of 1.66 ± 0. 25 （ P 〈 0. 05 ）. While HVEM mRNA expression did not change significantly （ P 〉 0. 05 ）. The 7th and 14th day after TC-1 cells inoculation, the average fluorescence intensity of BTLA expression on the surface of tumor infiltrating lymphocytes was 33. 5 and 51.8, respectively, in which there was statistically significant difference （ P 〈 0. 05 ） ; while the difference of HVEM expression was not statist

关 键 词：宫颈肿瘤 受体 免疫 HSP70热休克蛋白质类 疱疹病毒科 疾病模型 动物 

分 类 号：R737.33[医药卫生—肿瘤]