反转录病毒介导端粒酶基因对人口腔黏膜角化细胞寿命的影响

上海口腔医学 ›› 2014, Vol. 23 ›› Issue (3): 304-307.

反转录病毒介导端粒酶基因对人口腔黏膜角化细胞寿命的影响

黄吉燕^{1, 2}, 刘伟³, 周曾同¹, 周海文¹

1.上海交通大学医学院附属第九人民医院·口腔医学院口腔黏膜病科,上海市口腔医学重点实验室,上海 200011;
2.上海中医药大学附属曙光医院口腔科,上海 201203;
3.上海交通大学组织工程研究中心上海市组织工程重点实验室,上海 200011

收稿日期:2013-12-09 出版日期:2014-06-20 发布日期:2014-09-09
通讯作者: 周海文,Tel:021-23271699-5696,E-mail:haiwen39@hotmail.com
作者简介:黄吉燕(1976- ), 女, 硕士, 主治医师, E-mail:coucouh@163.com

The effect of retrovirus-mediated hTRT transfection into cultured oral keratinocytes

HUANG Ji-yan^1,2, LIU Wei³, ZHOU Zeng-tong¹, ZHOU Hai-wen¹

1.Department of Oral Medicine, Ninth People’s Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology. Shanghai 200011;
2. Department of Stomatology, Shuguang Hospital of Shanghai Chinese Medicine University. Shanghai 201203;
3.Tissue Engineering Research Center of Shanghai Jiao Tong University, Shanghai Key Laboratory of Tissue Engineering. Shanghai 200011, China

Received:2013-12-09 Online:2014-06-20 Published:2014-09-09

摘要/Abstract

摘要： 目的:以pBABE-tert重组反转录病毒为载体,介导端粒酶反转录酶基因(hTRT)转染体外培养的人口腔角化细胞,探讨端粒酶对细胞寿命的影响。方法:将含有hTRT的pBABE-tert重组反转录病毒载体扩增后,转染体外培养的人口腔角化细胞,挑选阳性克隆进行传代培养,以PCR-ELISA 和PCR-PAGE电泳检测端粒酶活性。结果:获得端粒酶活性稳定表达的口腔角化上皮细胞克隆,寿命可延长至8~9代。结论:转导端粒酶反转录酶基因的人口腔角化细胞寿命延长1倍,但不能永生化,说明人口腔上皮角化细胞的永生化是复杂和多点调控的过程,端粒酶活性增加是细胞永生化的关键但不是唯一原因。

关键词: 体外培养, 人口腔黏膜角化细胞, 端粒酶反转录酶, 反转录病毒, 基因转染

Abstract: PURPOSE: Human telomerase reverse transcriptase (hTRT) was transfected into cultured oral keratinocytes (OKC) mediated by pBABE-tert recombined retrovirus to investigate the effect on OKC lifespan. METHODS: pBABE-tert recombined retrovirus loaded with hTRT gene was amplified by transfected PT67 cells, and then transfected into cultured OKC in vitro. The positive clones of OKC were separated by puromycin and subcultured. Telomerase activity was analyzed by telomerase PCR-ELISA and PCR-PAGE. RESULTS: The hTRT positive clones of OKC showed telomerase expression, with extending lifespan to 8-9 passages. CONCLUSIONS: The hTRT transfected OKC can prolong doubly lifespan but not be immortalized, which indicates that cellular immortality mechanism is complicated and multi-controled. Telomerase activity is the key for cell immortalization but not the only impact factor.

Key words: Cell culture, Oral Keratinocytes, Recombined retrovirus, hTRT/hTERT, Gene transfection

中图分类号:

R781.5

黄吉燕, 刘伟, 周曾同, 周海文. 反转录病毒介导端粒酶基因对人口腔黏膜角化细胞寿命的影响[J]. 上海口腔医学, 2014, 23(3): 304-307.

HUANG Ji-yan, LIU Wei, ZHOU Zeng-tong, ZHOU Hai-wen. The effect of retrovirus-mediated hTRT transfection into cultured oral keratinocytes[J]. Shanghai Journal of Stomatology, 2014, 23(3): 304-307.

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