中文摘要：

背景与目的：肿瘤干细胞学说的提出为肿瘤治疗提供了新的靶点和方向,但肿瘤干细胞的分离纯化一直是个难题。本研究拟从人小细胞肺癌细胞株H446中分离并鉴定出具有干细胞特性的侧群（SP）细胞,研究其生物学特征,为肿瘤干细胞的分离纯化奠定基础。方法：采用荧光激活细胞分选（FACS）技术分选得到H446细胞中SP细胞和非侧群（NSP）细胞,并检测纯度。观察形成悬浮肿瘤细胞球的能力,采用逆转录-聚合酶链反应（RT-PCR）及荧光定量PCR检测这两种细胞亚群中CD133、ABCG2、NucleosteminmRNA水平。MTT法比较SP细胞、NSP细胞及未分选细胞体外增殖能力及耐药性差异,流式细胞仪检测体外分化能力,裸鼠成瘤实验检测体内成瘤能力。结果：荧光显微镜下H446细胞中Hoechst33342阴性细胞约为（5.1±0.2）%。流式细胞分选结果显示,H446中SP细胞比例为（6.3±0.1）%。SP细胞在无血清培养基中形成悬浮肿瘤细胞球的能力强于NSP细胞。CD133、ABCG2在SP细胞中的表达是NSP细胞的（21.60±0.26）倍、（7.10±0.14）倍,差异有统计学意义（P〈0.01）;Nucleostemin在SP细胞中的表达是非SP细胞的（1.02±0.08）倍,差异无统计学意义（P〉0.05）。SP细胞体外增殖能力及耐药存活能力均明显强于NSP细胞及未经分选的细胞（P〈0.01）;SP细胞在体外可分化为NSP细胞,但NSP细胞在体外不可分化为SP细胞;SP细胞在裸鼠体内具有较强的致瘤性。结论：人小细胞肺癌细胞株H446中存在具有肿瘤干细胞特性的SP细胞,CD133、ABCG2可能是人小细胞肺癌干细胞的分子标志物。

英文摘要：

Background and Objective： Recently,the theory of cancer stem cells （CSCs） has presented new targets and orientations for tumor therapy. The major difficulties in researching CSCs include their isolation and purification. The aim of this study is to identify and characterize the side population （SP） cells in small cell lung cancer （SCLC） cell line H446,which lays the foundation for the isolation and purification of CSCs. Methods： Fluorescence-activated cell sorting （FACS） was used to sort SP and non-SP （NSP） cells from H446. Both subgroups were cultivated to survey the capacity to form into suspended tumor cell spheres. Reverse transcription-polymerase chain reaction （RT-PCR） and real-time PCR were used to evaluate the expression levels of the mRNA of CD133,ABCG2,and nucleostemin in both subgroups. The capacity of proliferation and the differences in drug resistance of both subgroups and unsorted cells were tested by the MTT method. The differentiation ability of both subgroups was determined by FACS. Proliferation was determined by subcutaneous tumor formation in nude mice. Results： The percent of Hoechst 33342 negative cells was about （5.1±0.2）% in H446 by fluorescence microscopy. The percent of SP cells was （6.3±0.1）% by flow cytometry. SP cells had a stronger capability of forming into tumor spheres than NSP cells. The mRNA expression levels of ABCG2,CD133,and nucleostemin in SP cells were 21.60±0.26,7.10±0.14,and 1.02±0.08 folds higher than that in NSP cells （ P0.01,P0.01,and P0.05,respectively）. In vivo,SP cells showed better proliferative ability and tougher viability when treated with drugs. SP cells can differentiate into NSP cells,but NSP cells cannot differentiate into SP cells. SP cells had a greater ability to form tumors. Conclusions： The H446 cell line contained some SP cells with stem cell properties. CD133 and ABCG2 may be cancer stem cell markers of SCLC.