目的：从大细胞肺癌组织中分离、鉴定肺癌干细胞样细胞，为进一步研究靶向肺癌干细胞的免疫治疗奠定基础。方法：利用无血清培养基从大细胞肺癌组织中分离、培养肿瘤细胞并建立大细胞肺癌细胞系。流式细胞仪分选表达CD44和（或）CD90的不同肺癌细胞亚群，通过单细胞克隆形成实验、平板集落形成实验、细胞球形成实验及X射线敏感实验研究不同肺癌细胞亚群的生物学特性。结果：应用原代细胞培养技术成功地从大细胞肺癌组织中培养出高纯度的原代肿瘤细胞，命名为LC006细胞，其传代次数可达30代以上。流式细胞仪分析显示, LC006细胞中有1.0%的细胞CD44呈强阳性表达（CD44++）。相比于主群的CD44弱阳性（CD44＋）细胞，CD44++ LC006细胞具有较强的平板集落形成能力，可形成holoclone克隆和细胞球；而CD44＋LC006细胞不能形成holoclone克隆和细胞球。应用CD44和CD90抗体可将LC006细胞分为4个亚群，分别是 CD44＋CD90+（12.8%）、CD44＋CD90-(86.3%)、CD44++CD90+(0.4%)和CD44++CD90-(0.5%)细胞；相比于其他细胞亚群，CD44++CD90+LC006细胞形成细胞球的能力最强，对X射线的抵抗力也最强。结论：成功分离和鉴定的CD44++CD90+LC006细胞是大细胞肺癌的肿瘤干细胞样细胞。

To isolate and identify lung cancer stemlike cells from large cell lung cancer specimens, so as to lay a foundation for immunotherapy targeting lung cancer stem cells. Methods: Cancer cells were isolated and cultured from large cell lung cancer specimens using serumfree medium, and a large cell lung cancer cell line was established. Lung cancer subset cells expressing different degrees of CD44 and (or) CD90 were sorted by flow cytometry. Biologic characters of the different subsets were studied by single cell clone formation assay, plat colony formation assay, cell sphere formation assay, and Xray radiation sensitive assay. Results: Highly purified primary cancer cells were successfully cultured from large cell lung cancer specimens using primary cell culture technology, and the cells, named LC006, could be passaged for more than 30 generations. Flow cytometry results showed that 1.0% of LC006 cells expressed stronger CD44（CD44++LC006 cells）than the main population, which was weakly positive for CD44 (CD44+LC006 cells). CD44++LC006 cells had stronger colongy formation ability and could form holoclones and cell spheres; however, CD4+LC006 cells formed neither holoclones nor cell spheres, indicating that the CD44++LC006 cells contained cancer stem cells. When CD44 and CD90 antibodies were costained, LC006 cells could be individuated into 4 subsets, i.e. CD44+CD90+ (12.8%), CD44+CD90-(86.3%), CD44++CD90+(0.4%), and CD44++CD90-(0.5%) LC006 cells; CD44++CD90+LC006 cells showed strongest cell sphere formation ability and Xray resistant ability than the other three subsets. These results indicated that CD90 might be used as another cancer stemlike cell marker for large cell lung cancer. Conclusion: CD44++CD90+LC006 cells are successfully isolated and identified from large cell lung cancer, which may be the cancer stemlike cells of large cell lung cancer.

R734.2; Q279

河南省重点科技攻关资助项目（No. 0623033100）；河南省卫生厅重点资助项目（No. 200802015）