【摘要】 目的比较肿瘤浸润性淋巴细胞(TIL)各亚群体外特异性杀伤肿瘤功能,摸索TIL细胞表面受体(TCR互补决定区3(CDR3)基因体外扩增的方法,为寻找TIL细胞中肿瘤特异性抗原受体奠定前期实验基础。方法从肺癌肿瘤组织中分离出TIL细胞,利用CD8、PD-1抗体将TIL细胞分群,实时无标记细胞功能分析技术(real time cellular analysis,RTCA)分析比较各亚群细胞特异性杀伤活性。利用巢式RT-PCR的方法建立TCR VαCDR3区基因体外扩增的方法。结果 TIL细胞中CD3+CD8+(CTL)细胞亚群占75.7%,PD-1的表达约为29.7%; CD8+PD-1+TIL细胞亚群比CD8+PD-1-TIL细胞具有更强的体外杀伤原代肿瘤细胞功能;巢式RT-PCR成功扩增出TCR Vα1CDR3区基因片段。结论CD8+PD-1+TIL细胞亚群具有较强的特异性杀伤肿瘤活性;建立了通过巢式RT-PCR体外成功获取TCR所有CDR3区基因片段的实验条件。更多还原

【Abstract】 Objective To compare specific killing tumor effects of tumor infiltrating lymphocytes( TIL) subsets,and explore the experimental conditions of amplifiing the complementary determinant region 3( CDR3) gene of TIL cell surface receptor( TCR) in vitro to lay a foundation for searching informations of tumor specific antigens in TIL cells. Methods TIL cells were isolated from lung cancer tissues,and clustered by CD8 and PD-1 antibodies. Cell specific killing activity of each subgroup was analysis and compared by Real Time Cellular Analysis( RTCA). TCR Valpha CDR3 genes were amplified in vitro by nested RTPCR. Results CD3+CD8 +( CTL) subsets of TIL cells were accounted for 75. 7%,and the expression of PD-1 was about29. 7%; CD8+PD-1+TIL subset has stronger killing effect on primary cancer cells in vitro than CD8+PD-1-TIL; TCR Valpha1 CDR3 genes were successful amplified t by nested RT-PCR in vitro. Conclusion CD8+PD-1+TIL subset has stronger specific anti-tumor activity. The experimental conditions for obtaining all CDR3 gene from TIL TCR in vitro by nested RT-PCR were established.更多还原