转染人端粒酶反转录酶永生化成釉细胞瘤细胞的生物学特性  

Characterization of growth and proliferation in a telomerase-immortalized ameloblastoma cell line

作　　者：陶谦[1] 吕标[1] 乔彬[1] 郑超群[1] 陈之锋[1] 

机构地区：[1]中山大学光华口腔医学院·附属口腔医院口腔颌面外科中山大学口腔医学研究所,广州510055

出　　处：《中华口腔医学杂志》2009年第8期474-478,共5页Chinese Journal of Stomatology

基　　金：广州市科技计划项目（2008Z1-E191）

摘　　要：目的转染人端粒酶反转录酶（human telomerase reverse transenptase，hTERT）基因建立永生化成釉细胞瘤（ameloblastoma，AM）细胞株并观察其生物特性。方法构建hTERT基因反转录病毒载体pLXSN—neo—hTERT转染AM细胞，G418筛选后进行免疫细胞化学（immunocytochemistry，ICC）鉴定细胞来源、细胞形态学观察、细胞增殖动力学研究、细胞衰老β-半乳糖苷酶（senescence associated β galactosidase，SA—β—Gal）染色、反转录聚合酶链反应（RT—PCR）检测hTERT基因表达、端粒重复序列扩增法检测端粒酶活性。结果未转染AM细胞体外培养43d停止增殖，细胞群体倍增数（population doublings，PDL）为6；转染后AM细胞增殖活跃，目前PDL值已达到65；广谱细胞角蛋白阳性、波形蛋白阴性。与未转染细胞相比，转染后细胞形态较未转染细胞不规则；SA—β—Gal染色结果未见阳性衰老信号。RT—PCR检测hTERT mRNA在转染AM细胞中稳定表达，TRAP检测端粒酶活性相对未转染细胞呈高表达。结论转染hTERT基因的AM细胞呈现稳定增殖的永生化状态，为进一步研究AM提供良好的体外模型。Objective To establish and characterize the cell line of ameloblastoma （AM） by transfection with human telomerase reverse transcriptase（hTERT）. Methods Primary cultures of AM cells were infected with a retroviral vector encoding hTERT. Infected cells were selected and checked by immunocytochemistry（ICC）, in vitro proliferation, reverse transcriptase polymerase chain reaction （RT- PCR）, senescence associated βgalactosidase staining （SA-β-Gal staining）, telomerase activity assay. Results Compared to the uninfected cells, which arrested at the population doublings （PDL） of 6, the infected cells were more active in proliferation and reached 65 PDL to date. ICC confirmed the epithelial origin of the infected cells based on positive pan-cytokeratin and negative vimentin expression. There was no senescent signal in infected cells but not in uninfected cells, hTERT mRNA and telomerase activity were detected stably in infected cells. Conclusions The infected AM cells were immortalized after transfection with hTERT and can serve as a genetically defined model for AM study.

关 键 词：成釉细胞瘤 转染 永生化 

分 类 号：R686[医药卫生—骨科学]