高肺转移人源骨肉瘤细胞系的建立及鉴定  

Establishment and identification of human osteosarcoma cell line with high lung metastasis

作　　者：支文兰 常君丽[1] 胡少朴[1] 王晓波 赵福来 孙星媛 马小平 杨燕萍[1] ZHI Wenlan;CHANG Junli;HU Shaopu;WANG Xiaobo;ZHAO Fulai;SUN Xingyuan;MA Xiaoping;YANG Yanping(Longhua Hospital,Shanghai University of Traditional Chinese Medicine/Spine Institute,Shanghai University of Traditional Chinese Medicine/Key Laboratory of Theory and Therapy of Muscles and Bones,Ministry of Education of China,Shanghai 200032,China)

机构地区：[1]上海中医药大学附属龙华医院,上海中医药大学脊柱病研究所,教育部筋骨理论与治法重点实验室,上海200032

出　　处：《中国癌症防治杂志》2021年第4期394-399,共6页CHINESE JOURNAL OF ONCOLOGY PREVENTION AND TREATMENT

基　　金：国家重点研发计划项目(2018YFC1704300,2020YFE021600);国家自然科学基金项目(81973877,81674006)。

摘　　要：目的建立具有高肺转移能力的人源骨肉瘤细胞系,为探索骨肉瘤肺转移的发生机制提供研究模型。方法将GFP和荧光素酶标记的人源骨肉瘤143B细胞重悬到MEM培养基并与Matrigel胶按1∶1比例混匀,制成浓度为1×107/mL的细胞悬液。取10μL注射到4周龄BALB/c裸鼠的左侧胫骨内,建立胫骨内原位注射肺转移瘤模型(n=6)。收集肺转移结节制成单细胞悬液,在完全培养基中贴壁培养,并用新霉素(G418)抗性筛选后扩增,再次接种于裸鼠胫骨内;采用同样方法在体内循环2次后获得的细胞命名为143B-HLM。采用体外实时细胞迁移实验检测细胞迁移情况,RT-qPCR和Western blot检测迁移浸润关键分子标志物的表达情况,同时观察肺转移瘤模型肺部肿瘤结节的形成情况。结果与亲本人源骨肉瘤143B细胞相比,建立的143B-HLM细胞体外迁移能力增强,波形蛋白(Vimentin)和锌指转录因子(Slug)的蛋白表达量增加(均P<0.05),Vimentin、Slug、锌指转录抑制因子(Snail)和凋亡抑制基因(Survivin)的mRNA表达水平也升高(均P<0.05)。与注射143B细胞的裸鼠相比,裸鼠胫骨内原位注射143B-HLM细胞的荷瘤模型中,原位肿瘤体积减小(P<0.05),肺部肿瘤转移荧光信号显著增强,肺转移的裸鼠数目也更多(P<0.01)。结论骨肉瘤肺转移细胞经体内循环筛选及分离,成功建立了稳定的具有高肺转移能力的骨肉瘤细胞系143B-HLM。ObjectiveTo establish a human osteosarcoma cell line with high lung metastasis ability and provide a research model for explore the mechanism of lung metastasis of osteosarcoma.MethodsThe human osteosarcoma 143 B cells labeled with GFP and luciferase were resuspended into a MEM medium and mixed with Matrigel gel(1∶1)to make a cell suspension(1′107/mL),and 10μL of the suspension was injected into the left tibia of 4-week-old BALB/c nude mice to establish a model of lung metastasis by in situ injection into the tibia(n=6).The lung metastasis nodules were collected to make a single-cell suspension,cultured adherently in a complete medium,and amplified with neomycin(G418)resistance screening,then inoculated into the tibia of nude mice again.The cells obtained after circulating twice in vivo by the same method were named 143 B-HLM.The cell migration was detected by the vitro real-time cell migration assay,the expression of key molecular markers of cell migration and infiltration was detected by RT-qPCR and Western blot,and lung tumor nodule formation in lung metastatic tumor model was observed.ResultsCompared with parental human osteosarcoma143 B cells,143 B-HLM cells had higher migratory capacity in vitro;the protein expression of Vimentin and Slug,and the mRNA expression levels of Vimentin,Slug,Snail,and Survivin were increased(all P<0.05).Compared with nude mice injected with 143 B cells,the tumor-bearing model in which 143 B-HLM cells were injected in situ into the tibia of nude mice,the tumor volume in situ was decreased(P<0.05),the fluorescence intensity of lung tumor metastasis was significantly increased,and the number of lung metastasis was also increased(P<0.01).ConclusionsA stable osteosarcoma cell line 143 B-HLM with high lung metastatic capacity was successfully established through in vivo circulation screening and isolation of osteosarcoma metastatic cells.

关 键 词：骨肉瘤 143B-HLM 高肺转移细胞系 

分 类 号：R738[医药卫生—肿瘤]