增殖选择性腺病毒体外净化造血干细胞的研究  

In vitro purging the contaminating breast cancer cells from hematopoiefic stem cell grafts with reduplication-selective recombinant adenovirus

作　　者：战淑珺[1] 吴世凯[1] 宋三泰[1] 林晨[2] 张雪燕[2] 江泽飞[1] 胡放 

机构地区：[1]军事医学科学院附属307医院乳腺癌内科,北京100071 [2]中国医学科学院肿瘤研究所国家分子肿瘤实验室,100021 [3]上海三维生物技术有限公司,200001

出　　处：《临床肿瘤学杂志》2006年第4期254-258,共5页Chinese Clinical Oncology

基　　金：全军"十五"攻关课题(01QQ47)

摘　　要：目的：探讨增殖选择性腺病毒H101对造血干细胞体外净化的效果.方法：通过集落形成法、MTT比色法,观察H101对乳腺癌细胞及造血干细胞增殖的影响.应用RT-PCR检测CK-19mRNA表达,并结合肿瘤细胞集落形成法,建立H101净化效果的评价体系.在此基础上,评价H101模拟体外净化乳腺癌细胞和造血干细胞混合物的效果.结果：H101对乳腺癌细胞有显著的杀伤效应,在0～400MOI感染滴度范围内,存在明确的剂量效应关系,但H101对造血干细胞毒性较小,感染滴度达800MOI时对细胞增殖能力仍无明显影响.建立了RT-PCR及肿瘤细胞集落形成两种评价净化效率的方法,瘤细胞检测敏感度均可达6个对数级.模拟净化试验中,H101在感染滴度≥200MOI、混合培养体积1ml、混合培养时间2小时,可以达到5个对数级的乳腺癌细胞清除率,而对造血干细胞功能影响很小.结论：增殖选择性腺病毒可靶向净化造血干细胞中混有的乳腺癌细胞,为乳腺癌患者自体造血干细胞移植物的体外净化提供了新途径.Objective：In vitro, to develop a new method for purging the tumor cells from hematopoietic stem cell （HSC） products by using a reduplication-selective recombinant adenovirus H101. Methods： The cytopathic effects of H101 on breast cancer cell MDA-MB-231 and HSC were detected by MTr colorimetry and colony-forming uint （CFU） . An evaluating system to appraise the purging effect of H101 was set up by CFU-T and RT-PCR amplification of human cytokeratin-19 specific sequence. Finally, in vitro simulating clinical purging procedure, we observed the efficiency of the above mentioned purging method. Results： Proliferative activity of MDA-MB-231 decreased with the increasing dose of H101 in the range of 0- 400MOI. The clonogenic potential of the HSC was rarely affected by H101 even at 800MOI. A sensitive evaluating system was built up, by which 6 log level contamination of MDA-MB-231 in HSC was detected with RT-PCR and CFU-T. Following these studies, in vitro simulating purging procedure under optimal conditions, H101 induced 5 log level elimination of contaminating MDA-MB-231, while had little effect on the colonegenic forming potential of HSC. Conclusion：Co-incubation with H101 potentially provides a highly effective method for purging in vitro breast cancer cells from grafts prior to autologous transplantation.

关 键 词：增殖选择性腺病毒 乳腺癌 造血干细胞 体外净化 

分 类 号：R331.34[医药卫生—人体生理学] R733.7[医药卫生—基础医学]