摘要
背景:已有实验应用成分复杂的无血清培养基从骨肉瘤组织中分离出干细胞样肿瘤细胞。目的:应用简化的无血清培养基在U2-OS人骨肉瘤细胞系中分离培养出骨肉瘤干细胞并对其作肿瘤干细胞的相关检验。设计、时间及地点:干细胞体外培养实验,于2006-02/2007-02在兰州大学第二医院骨科研究所完成。材料:U2-OS人骨肉瘤细胞系购自中科院上海生命科学研究院细胞库。无血清培养基为在DMEM/F12培养基中添加重组表皮生长因子(20μg/L)和重组成纤维生长因子(20μg/L),L-谷氨酰胺(2mmol/L),胰岛素(4U/L),青霉素G(1×l05U/L),链霉素(100mg/L)。方法:取在含血清培养基中贴壁生长的U2-OS细胞,接种于含有表皮生长因子和成纤维生长因子的简化无血清培养基中,形成悬浮生长的骨肉瘤细胞球后观察其增殖、分化能力。用免疫细胞化学染色法检测间充质干细胞标志CD44、CD105蛋白在骨肉瘤细胞球中的表达。反转录-聚合酶链反应法检测U2-OS细胞及其肿瘤干细胞中胚胎多能干细胞管家基因Oct3/4mRNA的表达。主要观察指标:①悬浮细胞球的形成。②肿瘤干细胞增殖活性。③肿瘤干细胞的侵袭能力。④CD44、CD105蛋白在骨肉瘤细胞球细胞中的表达。⑤肿瘤干细胞中Oct3/4mRNA的表达。结果:U2-OS人骨肉瘤细胞系中有极少数的细胞能在简化无血清培养基中增殖形成骨肉瘤细胞球,具有很强的增殖分化能力,其表面具有间充质干细胞的膜分子CD44、CD105,表达胚胎干细胞管家基因Oct3/4mRNA。结论:U2-OS细胞系中存在肿瘤干细胞,简化无血清培养基可用于分离培养骨肉瘤干细胞。
BACKGROUND： Previous studies have used serum-free medium to isolate stem cells-like tumor cells from osteosarcoma tissues. OBJECTIVE： To isolate stem cells from human osteosarcoma cell line U2-OS with serum-free medium and to do relevant tests concerning cancer stem cells. DESIGN, TIME AND SETTING： The stem cell in vitro culture experiment was performed at the Institute of Orthopaedics, Second Hospital, Lanzhou University from February 2006 to February 2007. MATERIALS： U2-OS human osteosarcoma cell line was purchased from Cell Bank, Shanghai Institute of Life Science, Chinese Academy of Sciences. Serum-free medium was the DMEM/F12 medium, supplemented with recombinant epidermal growth factor （20 μg/L）, recombinant fibroblast growth factor （20μg/L）, L-glutamine （2 mmol/L）, insulin （4 U/L）, penicillin G （1 xl05 U/L） and streptomycin （100 mg/L）. METHODS： Adherent U2-OS cells in serum-supplemented medium were dissociated to single cell suspensions and were seeded in serum-free medium supplemented with epidermal growth factor and fibroblast growth factor. After the sarcospheres formed, proliferation and differentiation potentials were observed. Surface markers of mesenchymal stem cells （CD105 and CD44） were detected by immunocytochemistry. U2-OS cells and marker genes of pludpotent embryonic stem cells （Oct 314 mRNA） were detected by RT-PCR. MAIN OUTCOME MEASURES： Formation of suspended cell spheres; proliferation activity of cancer stem cells; invasion ability of cancer stem cells; CD44 and CD105 protein expression in osteosarcoma cell spheres; Oct3/4 mRNA expression in cancer stem cells. RESULTS： Osteosarcoma sarcospheres could be formed by a small subpopulation cells in U2-OS cell line. These cells were capable of self-renew and proliferation and expressed CD44 and CD 105 and OCt 3/4 m RNA. CONCLUSION： U2-OS cell line contains cancer stem cells that can be isolated with reducing serum-free medium.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2009年第19期3623-3627,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
甘肃省2006年科学事业费项目计划(QS061C3332)~~
