一种分离自体肿瘤细胞与TIL细胞新方法的建立.docVIP

一种分离自体肿瘤细胞与TIL细胞新方法的建立 邹征云 刘宝瑞 杜娟 钱晓萍 禹立霞 王立峰 殷海涛 南京大学医学院附属南京市鼓楼医院肿瘤科，江苏南京 210008 [摘要] 目的：寻求有效地从恶性胸腔积液和腹水中分离出较多的TIL细胞并保持其高度的增殖能力与抗瘤活性的方法。方法： 应用非连续密度梯度离心法与贴壁法对恶性胸腹水中的TIL进行分离，而后进行诱导培养，比较两种方法下TIL细胞的得率、增殖速度、杀瘤活性及培养前后的表型变化。结果： 贴壁法能够获得更多的TIL细胞，经统计学处理差异显著，P0.05；培养后两种方法所得TIL细胞的增殖速率、杀瘤活性及表型是一致的，经统计学处理差异无显著意义，P0.05。结论 ： 贴壁法具有操作简便、细胞得率高的优点，值得临床推广应用。 [关键词] 淋巴细胞，肿瘤浸润；离心法，梯密度；胸膜积液，恶性；腹水 [中图分类号] R730.51 A New Approach for Isolating Tumor Cells and Tumor Infiltrating Lymphocytes Zou Zheng-yun, Liu Bao-rui,Du Juan, et al. Department of Oncology, Nanjing Drum Tower Hospital, Faculty of Medicine, Nanjing University, Nanjing210008,China. Abstract: Objective To set up an efficient way to separate much more functional tumor infiltrating lymphocyte (TIL) from malignant pleura fluid and ascites. Methods Isolated by two ways, one being the discontinuous density gradient centrifugation, the other being the new attachment method we established, TILs were cultivated in the same conditions, and theirs proliferation as well as the killing activity and the phenotype changes were detected. Results It was obviously higher in quantity by the new attachment method compared with the way of discontinuous density gradient centrifugation; there was no difference in the proliferation or the killing activity in vitro as well as the phenotype changes between two ways. Conclusion The way of attachment is simple and high efficient for cells separation; it is deserved to be broadly applicated. Key Words: tumor infiltrating lymphocyte; isolation; pleura effusion; ascites 如何从恶性胸腔积液和腹水中分离出较多肿瘤细胞与TIL前体细胞？常规方法是采用淋巴细胞分离液进行非连续密度梯度离心法[1]。但是，这种方法烦琐，细胞丢失多，于是在实践中我们摸索并建立了一种比较简便、快捷而又有效的方法—贴壁法，该方法能从恶性胸腔积液和腹水中分离出较多的恶性肿瘤细胞及TIL前体细胞，有着极其便利的应用价值。 一、材料与方法 （一） 材料收集 1. 标本采集 收集本科2002年10月-2003年3月间6例晚期肿瘤患者（2例肺癌、1例肝癌、1例胃癌、1例十二指肠腺癌）的胸腔积液或腹水各200ml（肝素抗凝5U/ml），立即进行分离制备试验。 2. 主要试剂 淋巴细胞分离液由上海试剂二厂生产；RPMI1640培养基为美国GIBCOBRL公司产品；新生牛血清购自杭州四季青生物工程材料研究所；注射用人重组IL-2由北京四环药品厂生产；OKT3为古巴分子中心产品；DMSO及MTT均为Sigma公司分装；红细胞裂解液和缓冲液均用分析纯产品按常规方法配制