寡克隆肝癌浸润淋巴细胞的制备及其对自体癌细胞的杀伤活性  

Preparation of oligoclonal hepatocellular carcinoma-infiltrating lymphocytes and their cytotoxicity to autologous cancer cells

作　　者：张宗勤[1,2] 陈磊[2] 李鹏鹏[1,2] 张小峰[1] 孙斌[2] 钱海华[2] 施乐华[1] 殷正丰[2] 

机构地区：[1]第二军医大学东方肝胆外科医院肝外四科,上海200438 [2]第二军医大学东方肝胆外科医院东方肝胆外科研究所分子肿瘤实验室,上海200438

出　　处：《中国肿瘤生物治疗杂志》2013年第6期696-699,共4页Chinese Journal of Cancer Biotherapy

基　　金：国家自然科学基金资助项目(No.81101716;No.81272669);国家传染病重大专项课题资助项目(No.2012ZX10002012-10)~~

摘　　要：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 develop a novel method for isolation and culture of the oligoclonal tumor-infiltrating lymph- cytes （TILs） with a stronger cytotoxic activity to autologous hepatoma carcinoma cells. Methods： Fresh human hepatocel lular carcinoma （HCC） tissues were dissected to prepare conventional TILs and oligoelonal TILs by the way of enzymatic digestion with physical disaggregation and the new way of oligoclonal TILs culture from a single tumor fragment in micro cultures. After 2-week culture, the oligoclonal TILs with a stronger cytotoxic activity were mergered for further expending. Different TILs-mediated lysis of HCC cells in vitro was determined by eolorimetric tetrazolium （MTT） assay to compare the specific cytotoxicity between different oligoclonal TILs and between bulk TILs and oligoclonal TILs. Results： Lymphocytes could gradually migrate out of each tumor fragment and expand in the culture with high levels of IL-2. Oligoclonal TILs generated from different fragments showed different cytotoxic activities against autologous HCC cells, and the difference in cytotoxic activity between different oligoclonal TILs was significant （P 〈0.01 ）. Moreover, the cytotoxic activity of oligo clonal TILs was significantly stronger than that of bulk TILs （ [ 72.56 ± 6.69 ] % vs [ 46.24 ± 4. 03 ] %, P 〈 0.01 ）. Conclusion： The cytotoxic activity against autologous HCC cells of oligoclonal TILs generated from HCC tissues is nmchstronger than that of conventional TILs.

关 键 词：肿瘤浸润淋巴细胞 肝癌 寡克隆扩增 细胞毒活性 

分 类 号：R735.7[医药卫生—肿瘤] R730.51[医药卫生—临床医学]