基于荧光定量PCRDNA熔解曲线技术分析浸润性导管乳腺癌患者外周血和肿瘤组织TCR beta链CDR3谱系（英文）

TRBV gene expression in PBMCs and TILs in patients with infiltrating ductal breast carcinomas analyzed by a DNA melting curve( FQ-PCR) technique using TCR beta chain CDR3 spectratyping

目的采用TCR beta链CDR3谱系荧光定量PCR的DNA熔解曲线技术,探讨浸润性导管乳腺癌患者外周血T细胞和肿瘤组织T细胞之间TRBV基因表达情况。方法从遵义医学院附属医院甲乳外科采集乳腺癌志愿患者的乳腺肿瘤组织和外周血样本,选取病理诊断为浸润性导管癌的志愿患者23例,免疫组织化学检测ER、PR、PS2、C-erb B-2、nm23、P53和Ki-67的表达。分离每位志愿患者外周血单个核细胞(PBMCs),提取PBMCs和肿瘤部位组织总RNA,逆转录为c DNA,荧光定量PCR技术扩增各c DNA样本的TCR beta链CDR3区,DNA熔解曲线法对比分析各样本TRBV基因家族的表达情况。结果 23例浸润性导管乳腺癌志愿患者,外周血样本中TRBV6、TRBV8、TRBV12、TRBV14和TRBV24表达超过50%,肿瘤组织样本中TRBV6、TRBV12、TRBV14和TRBV24表达超过50%。选取的23例样本中的20例,其外周血和肿瘤组织均过表达相同的TRBV基因家族,但总TRBV基因家族的表达与ER、PR、PS2、C-erb B-2、nm2、P53和Ki-67的阳性或阴性没有明显的相关性。结论浸润性导管乳腺癌患者的外周血和肿瘤组织之间TRBV基因表达出现一致性,但表现出复杂多样性,与乳腺肿瘤相关抗原无明显相关,其机制可能与乳腺癌个体化的全身和局部免疫应答相关,对乳腺癌外周血和肿瘤组织部位特异T细胞TRBV基因表达探讨可为乳腺癌的免疫机制和个体化治疗提供理论基础。

Objective To probe TRBV gene expression in peripheral blood mononuclear cells（ PBMCs） and in tumor-infiltrating lymphocytes（ TILs） in patients with infiltrating ductal breast carcinomas（ IDCAs） using T cell receptor（ TCR） beta chain complementarity-determining region 3（ CDR3） spectratyping with a DNA melting curve and fluorogenic quantitative polymerase chain reaction（ FQ-PCR） technique. Methods Peripheral blood and tissue samples were obtained from 23 patients with IDCA tumors. Total RNA was extracted from PBMCs and tumor tissues and then reverse transcribed into c DNA. FQ-PCR was used to amplify the human TCR beta chain CDR3 region with primers for TRBV and TRBC genes. Results TCR beta CDR3 spectratyping showed preferential expression of some TRBV genes in the PBMCs and TILs of all 23 patients with IDCA tumors. TRBV6,TRBV8,TRBV12,TRBV14,and TRBV24 were expressed in greater than 50% of PBMCs; and TRBV6,TRBV12,TRBV14 and TRBV24 were expressed in greater than 50% of TILs. More than 80% of the patients（ 20 /23） overexpressed the same genes in both PBMCs and TILs. Patients with positive or negative tumor markers of estrogen receptor（ ER）,progesterone receptor（ PR）,PS2,C-erb B-2,nm23,P53 and Ki-67 had no significant common TRBV gene expression. Conclusion TRBV gene expression is complex and diverse in patients with IDCA tumors. TRBV gene expression may be attributable to the diversity of breast tumor antigens and the different immune responses observed in individual patients. Exploring the immunological mechanism of T cells may provide a basis for individual T cell-directed therapy for breast cancer.