人端粒酶催化亚基基因转染大鼠颅神经嵴干细胞向永生化细胞的转变  

Immortalization of rat cranial neural crest stem cells induced by human telomerase reverse transcriptase gene transfection

作　　者：吕红兵[1] 王捍国 吴甘茶[1] 闫福华[1] 

机构地区：[1]福建医科大学附属口腔医院牙体牙髓科,福建省福州市350002 [2]解放军第四军医大学口腔医院牙体牙髓科,陕西省西安市710032

出　　处：《中国组织工程研究与临床康复》2009年第23期4457-4461,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：教育部科学技术研究重点项目"大鼠颅神经嵴细胞的永生化及向成牙本质细胞的诱导分化"(JA06012)~~

摘　　要：背景：颅神经嵴干细胞可以作为颌面部各种组织和器官分化研究的重要细胞来源，如何使其获得永生化具有重要意义。人类细胞自发永生化的频率非常低，而一些永生化的基因可以显著增加这种频率。目的：观察人端粒酶催化亚基基因在大鼠颅神经嵴干细胞永生化过程中的作用。设计、时间及地点：观察性实验，于2007—09／2008-06在解放军第四军医大学完成。材料：选择清洁级孕8．5dSD大鼠3只，6周，体质量180g；先天性细胞免疫缺陷动物雄性Balb／c裸小鼠，体质量18～21g，均由解放军第四军医大学实验动物中心提供。质粒PCIneo-hTERT由解放军第四军医大学口腔医院牙体牙髓科提供。方法：原代培养大鼠颅神经嵴干细胞后，将含有人端粒酶催化亚基基因的质粒PCIneo-hTERT转入大鼠颅神经嵴干细胞。经G418筛选后扩增，并连续培养。采用免疫细胞化学法观察转染细胞内人端粒酶催化亚基基因和颅神经嵴干细胞的特异标志物P75的表达，检测细胞的端粒酶活性，绘制细胞生长曲线观察其增殖能力，并通过裸鼠移植实验了解转染细胞的特性。主要观察指标：细胞克隆、特异性蛋白P75表达、端粒酶活性、增殖能力及致瘤性实验结果。结果：①质粒PCIneo—hTERT转染细胞后24h，有少量细胞死亡。加G418后48h，细胞逐步开始大量死亡。12d后出现抗性细胞克隆，共有3个细胞克隆生长良好。分别命名为克隆1、2、3。克隆1和克隆2经过20～25代的传代后，细胞发生衰老、死亡：而克隆3在体外长期培养条件下生长状态良好，稳定表达人端粒酶催化亚基和P75。经转染后颅神经嵴干细胞的端粒酶活性明显升高，且能持续表达。②人端粒酶催化亚基基因转染后，3个细胞克隆在初期的增殖能力较强，随后克隆1、2的增殖速度逐渐变慢，出现死亡，克隆3越过衰老期，且无致瘤BACKGROUND： Cranial neural crest stem cells were the main source of cranio-facial tissue and organ. It is essential to immortalize these cells. The frequency of spontaneous immortalization of human cells is low, but some immortalization genes can significantly increase this frequency. OBJECTIVE： To investigate the effect of human telomerase reverse transcriptase （hTERT） in the immortalization of rat cranial neural crest stem cells. DESIGN, TIME AND SETTING： The observational experiment was performed at the Fourth Military Medical University of Chinese PLA from September 2007 to June 2008. MATERIALS： Three embryonic 8.5 d rats （6 weeks, 180 g）, and congenital cellular immune defects male Balb/c nude mice （18 21 g） were obtained from Experimental Animal Center of Fourth Military Medical University of Chinese PLA. Plasmid PCIneo-hTERT was supplied by Department of Endodontics, Stomatology Hospital, Fourth Military Medical University of Chinese PLA. METHODS： Primary rat cranial crest stem cells were cultured and transfected with eurayotic expressing plasmid PCIneo-hTERT encoding hTERT gene. Positive clones were selected by G418 and expanded for continuous culture. Expression of hTERT and P75 were detected in stable clone by immunocytochemical method. Telomerase activity was also measured. Growth curves were depicted to show proliferation of clones. Nude mice with transfected cells were used observe the biology features of cell clones. MAIN OUTCOME MEASURES： Cell clone, specific protein P75 expression, telomerase activity, proliferation, tumorigensis test were measured. RESULTS： Following 24 hours PCIneo-hTERT transfection, a few cells died. After adding G418 for 48 hours, cells gradually died in a large number. At day 12, cell clone appeared, and about 3 cell clones grew well, which were named clones 1, 2 and 3. Following 20-25 passages, clones 1 and 2 died. However, clone 3 grew well in vitro, and stably expressed hTERT and P75. Following transfection, telomerase activity obviously

关 键 词：颅神经嵴干细胞 永生化 转染 端粒酶催化亚基 

分 类 号：R394.2[医药卫生—医学遗传学]