摘要
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(magnetic 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。
Background and objective It has been proven that cancer stem cell existed in variety of cancer, which an significant difference 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 differences in CD133- positive cells and CD133-negative cells. The aim of this study is to isolate CD133＋ cells and CD133- cells from lung cancer cell line A549, explore 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, bF GF） in vitro. The colony formatting efficiency of CD133＋ cells, CD133- cells and cells without sorting was tested by colony-forming assay. The differentiation of sphere was induced by culturing in DMEM-F12 medium（containing serum）. The 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. The rates of CD133＋ cell colony formation（57.1%） was significantly higher than that of CD133- cells（3.3%）. Sphere（CD133＋/CK7-） was induced to differentiate, and CK7 expression was found in differentiated cells. The expression levels of 19 metastasis-related genes from CD133＋ cells and CD133- cells were significant different. Little CD133 positive cells which distributing around the cancer nests were found in lung cancer tissue. The expression of CD133 was not related to tumor types, cell differentiation or TNM stage. Conclusion CD133＋ cells exhibit the characteristics of cancer stem cells. The difference of metastasis-related gene expression levels was
出处
《中国肺癌杂志》
CAS
CSCD
北大核心
2015年第3期123-130,共8页
Chinese Journal of Lung Cancer
基金
教育部博士点基金博导类课题(No.20134423110001)
广东省自然科学基金(No.S2012010010181)
广州市科技计划项目(No.2014Y2-00171)
广州市教育系统创新学术团队项目(No.13C06)资助~~
关键词
CD133
肺腺癌
磁式分选
基因芯片
转移相关基因
CD133
Adenocarcinoma of lung
Magnetic activated cell sorting（MACS）
DNA microarray
Metastasis-related genes
