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目的:建立一种新的寡克隆肝癌浸润淋巴细胞(tumor infiltrating lymphocyte,TIL)分离培养方法,获得具有更强自体肝癌细胞杀伤活性的TIL。方法:以新鲜切除的人肝癌组织标本为材料,分别采用酶消化结合整块肝癌组织机械处理的传统方法和微小肝癌组织块培养的方法分离制备常规TIL和寡克隆TIL,培养2周后,将靶细胞毒活性较高的寡克隆TIL合并进一步培养扩增,采用MTT法分析不同寡克隆TIL对自体肝癌细胞的杀伤活性,比较不同寡克隆TIL之间、常规TIL和寡克隆TIL对自体肝癌细胞的杀伤活性。结果:在含有IL-2的培养体系中,TIL可自行从微小肝癌组织块中逐渐浸润出来并增殖。各个寡克隆TIL对自体肝癌细胞均有一定的杀伤活性,但是不同寡克隆TIL对于自体肝癌细胞的细胞毒活性有明显差异(P<0.01),寡克隆肝癌TIL对于自体肝癌细胞的细胞毒活性明显高于常规肝癌TIL[(72.56±6.69)%vs(46.24±4.03)%,P<0.01]。结论:寡克隆TIL分离培养方法制备的寡克隆肝癌TIL较常规TIL具有更强的自体肝癌细胞杀伤活性。
OBJECTIVE: To establish a new method of isolation and cultivation of tumor infiltrating lymphocytes (TILs) with oligoclonal liver cancer, and to obtain TIL with more self-hepatocarcinoma cell killing activity. Methods: Freshly resected human hepatocellular carcinoma tissue samples were used to prepare conventional TIL and oligoclonal TIL by using enzymatic digestion combined with mechanical treatment of whole liver cancer tissues and culturing of tiny liver cancer tissue pieces respectively. After culturing for 2 weeks, The oligoclonal TIL with higher target cytotoxicity was further cultured and expanded. The killing activity of different oligoclonal TILs on autologous hepatocarcinoma cells was analyzed by MTT assay. The effects of different oligoclonal TILs, conventional TILs and oligoclonal TILs on autologous hepatoma cells The killing activity. Results: In the culture system containing IL-2, TIL could gradually infiltrate and proliferate from the tiny liver cancer tissue mass. Each oligoclonal TIL had certain cytotoxic activity on autologous hepatocarcinoma cells, but the cytotoxic activity of different oligoclonal TIL on autologous hepatocarcinoma cells was significantly different (P <0.01). The cytotoxic activity of oligoclonal TIL on autologous hepatocarcinoma cells was significantly Which was higher than that of TIL [(72.56 ± 6.69)% vs (46.24 ± 4.03)%, P <0.01]. Conclusion: The oligoclonal TIL isolated from cultured TILs prepared by oligoclonal TIL has stronger self-hepatocarcinoma cell killing activity than conventional TIL.