Wang Bo, Yang Huan, Huang Yu-Zheng, Yan Ru-Hong, Liu Fen-Ju, Zhang Jun-Ning
Department of Radiotherapy, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215006, P. R. China.
Chin J Cancer. 2010 Mar;29(3):254-60. doi: 10.5732/cjc.009.10330.
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.
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.
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 (P < 0.01, P < 0.01, and P > 0.05, 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.
The H446 cell line contained some SP cells with stem cell properties. CD133 and ABCG2 may be cancer stem cell markers of SCLC.
近年来，癌症干细胞（CSCs）理论为肿瘤治疗提供了新的靶点和方向。研究CSCs的主要困难包括其分离和纯化。本研究旨在鉴定和表征小细胞肺癌（SCLC）细胞系H446中的侧群（SP）细胞，为CSCs的分离和纯化奠定基础。
采用荧光激活细胞分选（FACS）技术从H446中分离SP细胞和非SP（NSP）细胞。培养两个亚群以检测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）和实时PCR评估两个亚群中CD133、ABCG2和核干细胞因子mRNA的表达水平。采用MTT法检测两个亚群及未分选细胞的增殖能力和耐药性差异。通过FACS确定两个亚群的分化能力。通过裸鼠皮下肿瘤形成确定增殖情况。
荧光显微镜下，H446中Hoechst 33342阴性细胞百分比约为（5.1±0.2）%。流式细胞术检测SP细胞百分比为（6.3±0.1）%。SP细胞形成肿瘤球的能力强于NSP细胞。SP细胞中ABCG2、CD133和核干细胞因子的mRNA表达水平分别比NSP细胞高21.60±0.26、7.10±0.14和1.02±0.08倍（P分别<0.01、<0.01和>0.05）。在体内，SP细胞在药物处理时显示出更好的增殖能力和更强的活力。SP细胞可分化为NSP细胞，但NSP细胞不能分化为SP细胞。SP细胞具有更强的肿瘤形成能力。
H446细胞系含有一些具有干细胞特性的SP细胞。CD133和ABCG2可能是SCLC的癌症干细胞标志物。
