直肠癌肿瘤浸润性淋巴细胞中T细胞受体基因谱型特征

The characteristics of TCR repertoire in tumor-infiltrating lymphocytes from rectal cancer

目的 分析直肠癌肿瘤浸润性淋巴细胞 (tumor -infiltatinglymphocytes ,TIL)克隆的TCRβ基因谱型及CDR3区氨基酸序列 ,了解直肠癌TIL抗肿瘤细胞克隆的基因特征。方法 采用RT -PCR方法扩增直肠癌肿瘤组织、术前外周血和术后外周血T淋巴细胞TCTβ基因的 2 4个不同V基因片段 ,经过特殊的测序凝胶分离和银染色后 ,确定它们的TCRβ基因表达谱型。通过对PCR产物直接测序的方法 ,分析克隆性增殖的特征和CDR3区氨基酸序列。结果 直肠癌肿瘤组织TIL的TCRβ基因谱型中具有克隆性表达 ,表明直肠癌TIL是呈寡克隆性增生的淋巴细胞 ;来自 2例不同患者的肿瘤组织TIL中单克隆性表达的TCRβV11基因具有安全相同的DNA和氨基酸序列 ;其余四个寡克隆的CDR3区存在共同保守的氨基酸序列G/xGGN/xY(D)EQ ,这些序列可能直接接触直肠癌肿瘤组织相关抗原。结论 直肠癌肿瘤组织TIL细胞中存在特异性识别肿瘤相关抗原肽的T淋巴细胞克隆。

Objective: To study the anti-tumor clonal characteristics of tumor-infiltrating lymphocytes(TIL) in rectal cancer tissue, TCRβ gene repertoire and CDR3 amino acid sequence in TIL from the patients with rectal cancer were analyzed. Method: The 24 TCRβV gene from rectal cancer tissues, peripheral blood lymphocytes in pre-operation and post-operation were amplified by the method of combined reverse transcriptase-polymerase chain reaction (RT-PCR) with denaturation polyacrylamide sequencing gel eletrophoresis, Which is called TCRβ gene repertoire. In order to analyze the clonality of the over-expressing bands and the CDR3 sequence, the PCR reproduction was sequenced directly. Result: TCRβ gene repertoire in TIL of rectal cancer tissues was clonally expressed. The monoclonal expansion of TCRV11 from 2 different TIL had the same DNA sequence and amino acid sequence. The CDR3 in the rest of 4 oligoclones had a same conservative amino acid motif G/xGGN/xY(D)EQ. Conclusion: The clonal expanded cells in the TIL are supposed to be the peptide-specific anti-tumor T lymphocyte.