摘要
目的:采用无血清培养法从人骨肉瘤细胞株U2-OS中分离肿瘤干细胞,检测肿瘤干细胞特异性标志物Stro-1的表达。方法:实验于2006-02/2007-02在兰州大学第二医院骨科研究所完成。①实验材料:人骨肉瘤细胞株U2-OS由中国科学院细胞库提供;DMEM/F12(1∶1)培养基(Gibco公司);胎牛血清(杭州四季青公司);PCR引物由上海生物工程公司合成。②实验方法:取原代培养的骨肉瘤细胞经胰蛋白酶消化制备单细胞悬液,用含表皮生长因子20μg/L、碱性成纤维细胞生长因子20μg/L、L-谷氨酰胺2mmol/L、胰岛素4U/L、青霉素100U/mL和链霉素100U/mL,pH7.2～7.5的无血清DMEM/F12培养基重悬细胞,常规培养5～7d,待培养基中悬浮的肉瘤细胞球体积较大后,用无血清培养基重新吹打成单细胞悬液,按1∶2或1∶3比例传代。③实验评估:从上述骨肉瘤细胞株中分离出的肿瘤细胞球,用MTT法检测其增殖能力,免疫磁珠分选Stro-1阳性细胞,反转录-聚合酶链法检测Stro-1阳性细胞中Stro-1 mRNA的表达,免疫细胞化学染色的方法检测分化后成骨细胞特异性抗原的表达。结果:①肿瘤干细胞增殖活性:增殖潜伏期约为24h,传代后1～2d即可见肿瘤干细胞形成,以后体积逐渐增大。第7天吸光度值最高,与0d吸光度值比较差异有显著性意义(P<0.01)。②免疫磁珠法分选Stro-1阳性细胞:分选的Stro-1+细胞接种于无血清培养基后,24～48h即可形成和原代肿瘤干细胞球形态一样的干细胞球,而Stro-1-细胞却不能形成肿瘤细胞球。③骨肉瘤干细胞中Stro-1mRNA的表达:Stro-1+细胞和Stro-1-细胞mRNA扩增产物的吸光度值二者比较差异有显著性意义(P<0.05)。④肿瘤干细胞分化:分化2周的肿瘤干细胞骨形态蛋白及Ⅰ型胶原酶均呈阳性表达。结论:骨肉瘤细胞株中存在骨肉瘤干细胞,并具有自我更新和多向分化的能力。
AIM： To isolate cancer stem cells from human osteosarcoma cell line U2-OS with serum-free incubation techniques, and detect the expression of specific surface markers Stro-1 of cancer stem cells. METHODS： The experiment was conducted in the Institute of Orthopedics, Second Hospital of Lanzhou University between February 2006 and February 2007. ①Human osteosarcoma cell line U2-OS was obtained from cell bank of Chinese Academy of Science. DMEM/F12 （1：1）medium and fetal bovine serum （FBS） were purchased from Gibco Corporation and Hangzhou Sijiqing Company, respectively. The PCR primers were composed by Shanghai Bioengineering Company. ②The primary cultured cells of osteosarcoma were digested by Trypsin to prepare a single-cell suspension. The depositional cells were prepared to single-cell with serum-free DMEM/F12 medium containing epidermal growth factor （EGF） 20 μg/L, basic fibroblast growth factor （bFGF） 20 μg/L, L-Glutamine 2 mmol/L, insulin 4 U/L, penicillin G 100 U/mL and streptomycin 100 U/mL with pH of 7.2-7.5. After conventional culture for 5 to 7 days, colony volume became larger. The cell suspension was centrifuged, and were subcultured in serum-free medium according to 1：2 or 1：3. ③The proliferative capability of sarcospheres from human osteosarcoma cell line was estimated by MTT method. Stro-1 cells were isolated by immunomagnetic beads. Reverse transcription-polymerase chain reaction （RT-PCR） method was used to detect the expression of Stro-1^＋ mRNA in Stro-1^＋ cells. Expression of specific proteins of bone cells were detected by immunocytochemical stain. RESULTS： ①Proliferation of cancer stem cells： The delitescence of proliferation was 24 hours. The cancer stem cells formed sarcospheres after subcultured for 1 to 2 days, and the volume of sarcospheres was enlarged gradually. The highest absorbance （A） was measured at day 7. There were significant differences between A at day 0 and day 7 （P 〈 0.01 ）. ②Stro-1^＋ cells isolated by immunomagne
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第24期4706-4709,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
甘肃省科技厅基金资助(QSO61C3332)~~
