转导hTERT基因致人牙髓干细胞永生化的研究

上海口腔医学 ›› 2015, Vol. 24 ›› Issue (2): 135-140.

转导hTERT基因致人牙髓干细胞永生化的研究

高丽¹,王玉霞¹,江文欣¹,何智妍²,朱彩莲²,马瑞¹

1.上海交通大学医学院附属第九人民医院·口腔医学院牙体牙髓病科,上海 200011;
2.上海市口腔医学重点实验室,上海 200011

收稿日期:2014-08-14 出版日期:2015-04-20 发布日期:2015-07-24
通讯作者: 马瑞,Tel:021-23271699-5201,E-mail:marui1723@sina.cn
作者简介:高丽（1986-）,女,在读硕士研究生,E-mail:1127239423@sjtu.edu.cn
基金资助:
国家自然科学基金（81371143）; 上海市启明星计划（12QH1401400）

Immortalization of human dental pulp stem cells caused by transferring hTERT gene

GAO Li¹,WANG Yu-xia¹,JIANG Wen-xin¹,HE Zhi-yan²,ZHU Cai-lian²,MA Rui¹

1.Department of Endodontics, Shanghai Ninth People’s Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine. Shanghai 200011;
2. Shanghai Key Laboratory of Stomatology. Shanghai 200011, China

Received:2014-08-14 Online:2015-04-20 Published:2015-07-24
Supported by:
National Natural Science Foundation of China(81070826/81371143) and Shanghai Rising-Star Program (12QH1401400)

摘要/Abstract

摘要： 目的建立永生化人牙髓干细胞系（human dental pulp stem cell,hDPSC）,供口腔医学临床和基础研究使用。方法原代培养人牙髓细胞并筛选牙髓干细胞,以慢病毒为载体,导入端粒酶反转录酶基因（hTERT）全长cDNA,经筛选得到阳性克隆。体外连续传代,应用反转录PCR（reverse transcription PCR ,RT-PCR)和蛋白质印迹技术（Western blot）检测hTERT的整合和表达情况。结果成功地将hTERT基因转入人牙髓干细胞,得到的转化细胞端粒酶表达阳性,增殖旺盛,至今已传至第35代。结论转导外源性hTERT基因可以导致人牙髓干细胞永生化。

关键词: 端粒酶反转录酶基因, 牙髓干细胞, 永生化

Abstract: PURPOSE: To establish an immortalized human dental pulp stem cell line used for basic and clinical research of oral science. METHODS: Human telomerase reverse transcriptase (hTERT) cDNA was transferred into human dental pulp stem cells (hDPSCs) by lentivirus. The resultant stable clones reproduced successively and the expression of hTERT was identified. RESULTS: The hTERT gene was transferred into human dental pulp stem cells successfully. The transformed cells expressed telomerase activity and divided vigorously. p35 had been obtained so far. CONCLUSIONS: The hDPSCs can be immortalized by transferring exogenous hTERT gene to constitute telomerase activity.

Key words: Human telomerase reverse transcriptase, Dental pulp stem cell, Immortalization

中图分类号:

R780.2

高丽,王玉霞,江文欣,何智妍,朱彩莲,马瑞. 转导hTERT基因致人牙髓干细胞永生化的研究[J]. 上海口腔医学, 2015, 24(2): 135-140.

GAO Li,WANG Yu-xia,JIANG Wen-xin,HE Zhi-yan,ZHU Cai-lian,MA Rui. Immortalization of human dental pulp stem cells caused by transferring hTERT gene[J]. Shanghai Journal of Stomatology, 2015, 24(2): 135-140.

参考文献

[1] Simonsen JL, Rosada C, Serakinci N, et al. Telomerase expression extends the proliferative life-span and maintains the osteogenic potential of human bone marrow stromal cells [J]. Nat Biotechnol, 2002, 20(6): 592-596.
[2] Hilkens P, Gervois P, Fanton Y, et al. Effect of isolation methodology on stem cell properties and multilineage differentiation potential of human dental pulp stem cells [J]. Cell Tissue Res, 2013, 353(1): 65-78.
[3] Lin CS, Xin ZC, Dai J, et al. Commonly used mesenchymal stem cell markers and tracking labels: limitations and challenges [J]. Histol Histopathol, 2013, 28(9): 1109-1116.
[4] Struys T, Moreels M, Martens W, et al. Ultrastructural and immunocytochemical analysis of multilineage differentiated human dental pulp- and umbilical cord-derived mesenchymal stem cells [J]. Cells Tissues Organs, 2011, 193(6): 366-378.
[5] 李景辉, 张方明, 张振庭. 人恒牙牙髓干细胞体外定向诱导分化为成骨细胞的研究 [J]. 北京口腔医学, 2011, 19(3): 139-143.
[6] 李景辉, 张方明, 张振庭. 人恒牙牙髓干细胞分化为脂肪细胞的体外实验研究 [J]. 北京口腔医学, 2012, 20(3): 142-146.
[7] Yang GB, Li XY, Yuan GH, et al. Immortalization and characterization of human dental papilla cells with odontoblastic differentiation [J]. Int Endod J, 2013, 46(6): 565-572.
[8] Gronthos S, Mankani M, Brahim J, et al. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo [J]. Proc Natl Acad Sci USA, 2000, 97(25): 13625-13630.
[9] Bodnar AG, Ouellette M, Frolkis M,et a1. Extension of life-span by introduction of telomerase into normal human cells[J]. Science, 1998, 279(5349): 349-352.
[10] Counter CM, Hahn WC, Wei W, et al. Dissociation among in vitro telomerase activity, telomere maintenance, and cellular immortalization [J]. Proc Natl Acad Sci USA, 1998, 95(25): 14723-14728.
[11] Zhao CF, Hu HY, Meng L, et al. Immortalization of bovine mammary epithelial cells alone by human telomerase reverse transcriptase [J]. Cell Biol Int, 2010, 34(6): 579-586.
[12] Ouellette MM, McDaniel LD, Wright WE, et al. The establishment of telomerase-immortalized cell lines representing human chromosome instability syndromes[J]. Hum Mol Genet, 2000, 9(3): 403-411.

[1]	王静, 徐娜, 任慧迪. TNF-α通过ERK1/2-Runx2信号通路调控SHED成骨分化能力的实验研究[J]. 上海口腔医学, 2024, 33(2): 135-140.
[2]	刘岩, 单丹妮, 孙晶, 邹雨希, 袁长永. Let-7a对人牙髓干细胞增殖、凋亡和成骨分化的影响[J]. 上海口腔医学, 2023, 32(5): 468-474.
[3]	李宗谕, 李晓琳, 尉鹏功, 曲柳, 仇丽鸿, 于雅琼. 白藜芦醇在人牙髓干细胞成牙本质向分化中的调控机制研究[J]. 上海口腔医学, 2023, 32(2): 132-136.
[4]	付洪海, 孙乐刚, 丁昌成, 马向瑞, 黄玉梅. miR-31-5p对牙髓干细胞HIF-1α/BNIP3信号通路及成骨相关因子表达的影响[J]. 上海口腔医学, 2022, 31(3): 237-242.
[5]	李晓琰, 郑蕾, 马鹏涛, 张彦喜. 釉质基质蛋白通过miR-32对乳牙牙髓干细胞成骨与成脂分化的影响[J]. 上海口腔医学, 2021, 30(4): 367-373.
[6]	杨峥, 赵敏, 肖琳, 陈思萍. 不同低氧浓度对体外培养人牙髓干细胞生物学特性的影响[J]. 上海口腔医学, 2021, 30(3): 247-252.
[7]	马春燕, 潘清, 何磊, 杨海兵. 细胞外基质PDMS硬度对牙髓干细胞增殖和成骨分化的影响[J]. 上海口腔医学, 2021, 30(3): 253-257.
[8]	张维华, 於丽明, 韩欣欣, 潘杰, 邓佳佳, 朱露莹, 刘月华. 人牙髓干细胞条件培养基通过AMPK/PGC-1α途径改善氯化钴诱导的颏舌肌成肌细胞低氧损伤[J]. 上海口腔医学, 2020, 29(6): 573-579.
[9]	牛晨光, 李佳洋, 於丽明, 陈栋, 韦晓玲. 牡荆素对脂多糖诱导牙髓干细胞表达炎症因子的影响[J]. 上海口腔医学, 2020, 29(5): 462-465.
[10]	李月玲, 杨佳音, 徐睿. 牙髓干细胞对皮肤成纤维细胞衰老及增殖能力的影响[J]. 上海口腔医学, 2020, 29(5): 466-470.
[11]	常思佳, 杨柳, 李冉. 明胶-羟基磷灰石-磷酸三钙支架对hDPSCs的成骨刺激作用[J]. 上海口腔医学, 2020, 29(5): 492-498.
[12]	李静, 张艺, 王金华. 骨形态发生蛋白-2、-4、-6、-7和-9差异性介导永生化成牙本质细胞成骨分化[J]. 上海口腔医学, 2020, 29(3): 242-249.
[13]	王宇琛, 王辰, 刘娜, 蔡川, 李伟, 徐璐璐. TNF-α对人脱落乳牙牙髓干细胞促破骨细胞形成能力的影响[J]. 上海口腔医学, 2018, 27(5): 449-454.
[14]	卢皙, 刘上峰, 赵守亮. GLUD1基因对人牙髓干细胞的体外增殖、分化和矿化的影响[J]. 上海口腔医学, 2017, 26(4): 358-362.
[15]	李晓明, 袁长永, 朱绍跃, 刘宗响, 王鹏来. 基因转染EphB4对脱落乳牙牙髓干细胞成骨分化的影响[J]. 上海口腔医学, 2017, 26(4): 399-403.