CD133阳性/阴性肺癌细胞的分选、鉴定及差异基因的筛选

摘要

Background and objective It has been proven that cancer stem cell existed in variety of cancer, which an significant dierence of biological characteristics was observed between the cancer stem cells and non-cancer stem cells. And CD133 is considered to be cancer stem cell marker. So there may be significant dierences in CD133- positive cells and CD133-negative cells. e aim of this study is to isolate CD133+ cells and CD133- cells from lung cancer cell line A549, ex-plore their biological characteristics and screen the metastasis-related genes. Methods MACS was applied to isolate CD133+cells and CD133- cells from human lung cancer cell line A549. To observe the formation of sphere, CD133+ cells and CD133-cells were cultured in serum-free DMEM-F12 medium (containing EGF, bFGF) in vitro. e colony formaing eciency of CD133+ cells, CD133- cells and cells without sorting was tested by colony-forming assay. e dierentiation of sphere was in-duced by culturing in DMEM-F12 medium (containing serum). e metastasis-related genes (84 genes) of CD133+ cells and CD133- cells were detected by using DNA microarray. Immunohistochemistry was used to detect the expression of CD133 protein in Human lung cancer tissue. Results CD133+ cells formed sphere in serum-free DMEM-F12 medium,while the CD133- cells failed to form sphere. e rates of CD133+ cell colony formation (57.1%) was significantly higher than that of CD133- cells (3.3%). Sphere (CD133+/CK7-) was induced to dierentiate, and CK7 expression was found in dierentiated cells. e expression levels of 19 metastasis-related genes from CD133+ cells and CD133- cells were significant dierent. Lile CD133 positive cells which distributing around the cancer nests were found in lung cancer tissue. e expression of CD133 was not related to tumor types, cell dierentiation or TNM stage. Conclusion CD133+ cells exhibit the characteristics of can-cer stem cells. e dierence of metastasis-related gene expression levels was discovered between CD133+ cells and CD133-cells. CD82 plays an important role in mechanism of tumor metastasis.%背景与目的研究表明多种实体肿瘤中存在肿瘤干细胞（cancer stem cells, CSCs），肿瘤干细胞与非肿瘤干细胞的生物学特性存在已有的明显差异，而CD133被认为是肿瘤干细胞的标记物，因此CD133阳性细胞与CD133阴性细胞可能存在明显差异。本研究通过分选人肺腺癌A549细胞中的CD133阳性及CD133阴性细胞，鉴定两组细胞的生物学特性并在人组织标本进行验证，利用基因芯片筛选转移相关差异基因。方法采用磁式分选（mag-netic activated cell sorting, MACS）的方法对人肺腺癌A549中CD133阳性细胞、CD133阴性细胞进行分选，通过无血清条件培养、平板克隆、血清诱导分化及基因芯片等实验，比较两组细胞“sphere”形成、细胞增殖、细胞分化以及差异基因，并在人组织标本进行CD133表达与临床特性的验证分析。结果 CD133阳性细胞能在无血清培养基中悬浮生长并形成“sphere”；其平均克隆形成率（57.1%）明显高于CD133阴性细胞（3.3%）；CD133阳性细胞能被血清诱导分化、表达腺癌标志物CK7；两组细胞中的19个转移基因表达水平相差两倍或以上，差异最高达12倍；肺癌组织中CD133阳性细胞主要分布于癌巢周边，数量稀少，其表达与肿瘤组织学分型、分级及临床分期无关。结论 CD133阳性肺腺癌A549细胞具有CSCs特性；两组细胞在转移相关基因的表达存在明显差异，其中CD82可能在CD133阳性细胞的转移机制中起重要作用。