肝癌肿瘤浸润淋巴细胞_TIL_体外扩增及特性研究.docxVIP

中国细胞生物学学报 Chinese Journal of Cell Biology 2014, 36(7): 970–975DOI: 10.11844/cjcb.2014.07.0367 临床床细细胞胞生生物物学学 肝癌肿瘤浸润淋巴细胞(TIL)体外扩增及特性研究江珊珊唐艳潘科翁德胜赵靖靖何佳夏建川*(中山大学附属肿瘤医院生物治疗中心, 广州 510060)摘要 探讨从原发性肝癌(hepatocellular carcinoma, HCC)患者的术后肿瘤组织中分离肿瘤浸 润性淋巴细胞(tumor-infiltrating lymphocyte, TIL), 并进行体外大量扩增的方法。在该研究工作中, 组 织标本来源于术后的肿瘤癌旁组织, 经过组织破碎、酶消化后, 采用不连续密度梯度离心分离其中 淋巴细胞。分离的淋巴细胞先采用大剂量重组人白介素2(IL-2)(2 000 U/mL)进行引发, 然后采用同 种异体的外周血单核细胞作为饲养细胞进行扩增。针对9例原发性肝癌术后标本, 所分离的TIL均能 成功进行培养扩增, 扩增后细胞数为(1~5.5)×109。扩增倍数为111~572。扩增后的TIL, 表型检测发 现CD3+细胞的比例为(90.3±9.4)%, CD3+CD4+细胞的比例为(24.9±14.1)%, CD3+CD8+细胞的比例为(56.4±20.2)%, CD3+CD56+细胞的比例为(14.8±12.6)%。杀伤检测结果显示, 肝癌TIL对非自体肿 瘤细胞系HepG2和Bel-7402有较强的杀伤作用。因此, 采用该改良的方法, 原发性肝癌的TIL在体外 可以成功进行培养扩增, 并具有较强抗肿瘤活性, 可以作为肝癌术后巩固性免疫治疗的一种手段。关键词肝癌; 肿瘤浸润性淋巴细胞; 免疫治疗; 体外扩增培养In Vitro Culturing and Characteristics of Tumor-in fi ltrating Lympho cyte of Hepatocellul ar CarcinomaJiang Shanshan, Tang Yan, Pan Ke, Weng Desheng, Zhao Jingjing, He Jia, Xia Jianchuan*(Department of Biotherapy Center, Sun Yat-sen University Cancer Center, Guangzhou 510060, China)Abstract The aim was to investigate the methods of ex vivo expansion of tumor-infiltrating lympho- cyte (TIL) obtained from hepatocellular carcinoma (HCC) biopsy specimens with a rapid expansion protocol. In this study, the specimen of primary liver cancer came from adjacent carcinoma tissues. After crushing and enzy- matic digestion, the lymphocytes in the single-cell slurry were separated by density centrifugation. The isolated lymphocytes were triggered with high dose of recombinant human interleukin 2 (IL-2) (2 000 U/mL), then were expanded in large-scale using peripheral blood allogeneic mononuclear cells as feeder cells. Nine primary HCC samples were used to try to isolate and culture the TIL in vitro. After two-step culturing, the number of TIL could achieve (1.0~5.5)×109 cells, and the expansion fold was 111 to 572. After expansion, the ratio of CD3+ T cells was (90.3±9.4)%, the ratio of CD3+CD4+ T cells was (24.9±14.1)%, the