肝癌肿瘤浸润淋巴细胞（TIL）体外扩增及特性研究

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）×10^9。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。

In Vitro Culturing and Characteristics of Tumor-infiltrating Lymphocyte of Hepatocellular Carcinoma

The aim was to investigate the methods of ex vivo expansion of tumor-infiltrating lymphocyte （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 enzymatic 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）×10^9 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 ratio of CD3＋CD8＋ was （56.4±20.2）%, and the ratio of CD3＋CD56＋ was （14.8±12.6）%. Moreover, the TIL showed strong killing activity to HepG2 and Bel-7402 cell lines in vitro. Taken together, the TIL from hepatocellular carcinoma can be successfully cultured and expanded in vitro, and with strong anti-tumor activity. It can be served as a means of the candidate immunotherapy for postoperative HCC patients.