DLK2基因在胚胎软骨发育中的表达及意义

中国口腔颌面外科杂志 ›› 2016, Vol. 14 ›› Issue (4): 302-307.

DLK2基因在胚胎软骨发育中的表达及意义

徐伟峰*, 沈佩*, 范宝婷, 焦子先, 李慧萍, 张善勇

上海交通大学医学院附属第九人民医院·
口腔医学院口腔外科, 上海市口腔医学重点实验室,上海 200011

出版日期:2016-08-20 发布日期:2016-12-08
通讯作者: 张善勇,E-mail:zhangshanyong@126.com
作者简介:徐伟峰（1989-）,男,在读硕士研究生,E-mail:xwf19891215@163.com;沈佩（1987-）,女,硕士,住院医师,E-mail:shenpei1215@163.com。*并列第一作者
基金资助:
国家自然科学基金（81371168）

Expression and significance of DLK2 gene during cartilage development

XU Wei-feng, SHEN Pei, FAN Bao-ting, JIAO Zi-xian, LI Hui-ping, ZHANG Shan-yong

Department of Oral Surgery, Shanghai Ninth People's Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine,Shanghai Key Laboratory of Stomatology. Shanghai 200011, China

Online:2016-08-20 Published:2016-12-08

摘要/Abstract

摘要： 目的研究DLK2 (delta drosophila homolog-like 2 或delta like 2 homologue)在胚胎软骨发育中的表达及意义。方法检测ATDC5细胞系正常诱导分化过程中DLK2的表达改变;建立ATDC5-DLK2慢病毒过表达细胞系,检测DLK2基因对ATDC5细胞增殖及分化的影响;通过免疫组织化学检测DLK2在C57BL/6小鼠胚胎(E11.5 d,E16.5 d,E18.5 d）和出生后（P7 d和P14 d）膝关节的时空表达,采用SPSS16.0软件包对数据进行配对t检验。结果 ATDC5细胞正常诱导分化过程中,DLK2 的mRNA和蛋白水平逐渐下降;过表达DLK2后,ATDC5细胞增殖速率显著提高（P<0.05）,但ATDC5细胞成软骨标志物（Col2a1、Acan、Col10a1）的mRNA水平显著下降（P<0.05）;胚胎发育早期（E11.5 d）,DLK2在软骨形成处开始有较弱表达,在软骨发育过程中,其表达逐渐增加,而且DLK2在未成熟和成熟的软骨细胞均有表达,关节软骨表层的DLK2表达逐渐下降（P<0.05）,但深层几乎不变（P>0.05）。结论 DLK2可能是参与胚胎软骨发育过程的一个重要细胞因子。

关键词: DLK2, ATDC5细胞, 软骨发育

Abstract: PURPOSE: To study the expression and significance of DLK2 during embryonic cartilage development. METHODS: The expression of DLK2 was detected during the differentiation of ATDC5 cells. ATDC5-DLK2 cells which stably overexpressed DLK2 was constructed by lentivirus transfection, and the effect of DLK2 on the proliferation and differentiation of ATDC5 cells was studied. The temporal and spatial expression of DLK2 in cartilage of C57BL/6 mouse at embryos(E11.5,E16.5,E18.5 days) and postnatal time (P7 and P14 day) was examined by immunohistochemistry. The data was analyzed by paired t test using SPSS16.0 software package. RESULTS: The mRNA and protein levels of DLK2 decreased during the differentiation of ATDC5 cells; ATDC5-DLK2 cell line which stably ovexpressed DLK2 was established and identified, and DLK2 overexpression promoted the proliferation of ATDC5 cells (P<0.05),but significantly inhibited the expression of chondrogenic marker genes (col2a1, acan, col10a1)(P<0.05). DLK2 showed a weak expression in cartilage of early embryo (E11.5 days), while gradually increased during the development of cartilage. DLK2 was expressed in both immature and mature chondrocytes, but the expression of DLK2 in articular cartilage surface decreased (P<0.05), while almost unchanged in the deep of articular cartilage (P>0.05). CONCLUSIONS: DLK2 may be an important factor involved in cartilage development.

Key words: DLK2, ATDC5 cells, Cartilage development

中图分类号:

R786.2

徐伟峰*, 沈佩*, 范宝婷, 焦子先, 李慧萍, 张善勇. DLK2基因在胚胎软骨发育中的表达及意义[J]. 中国口腔颌面外科杂志, 2016, 14(4): 302-307.

XU Wei-feng, SHEN Pei, FAN Bao-ting, JIAO Zi-xian, LI Hui-ping, ZHANG Shan-yong. Expression and significance of DLK2 gene during cartilage development[J]. China J Oral Maxillofac Surg, 2016, 14(4): 302-307.

参考文献

[1] Nueda ML, Baladron V, Garcia-Ramirez JJ, et al. The novel gene EGFL9/ DLK2, highly homologous to DLK1, functions as a modulator of adipogenesis [J]. J Mol Biol, 2007, 367(5): 1270-1280.
[2] Rivero S, Ruiz-Garcia A, Diaz-Guerra MJ, et al. Characterization of a proximal Sp1 response element in the mouse DLK2 gene promoter [J].BMC Mol Biol, 2011, 12: 52.
[3] Smas CM, Sul HS. Pref-1,a protein containing egf-like repeats, inhibits adipocyte differentiation [J]. Cell, 1993, 73(4): 725-734.
[4] Lee K, Villena JA, Moon YS, et al. Inhibition of adipogenesis and development of glucose intolerance by soluble preadipocyte factor-1 (Pref-1)[J].J Clin Invest, 2003, 111(4): 453-461.
[5] Wang Y, Sul HS. Pref-1 regulates mesenchymal cell commitment and differentiation through Sox9 [J]. Cell Metab, 2009, 9(3): 287-302.
[6] Chen L, Qanie D, Jafari A, et al. Delta-like 1/fetal antigen-1(DLK1/ FA1)is a novel regulator of chondrogenic cell differentiation via inhibition of the Akt kinase-dependent pathway [J]. J Biol Chem, 2011, 286(37): 32140-32149.
[7] Abdallah BM, Jensen CH, Gutierrez G, et al. Regulation of human skeletal stem cells differentiation by DLK1/Pref-1 [J].J Bone Miner Res, 2004, 19(5): 841-852.
[8] Khan QE, Sehic A, Skalleberg N, et al. Expression of delta-like 1 homologue and insulin-like growth factor 2 through epigenetic regulation of the genes during development of mouse molar [J].Eur J Oral Sci, 2012, 120(4): 292-302.
[9] Pan RL, Wang P, Xiang LX, et al. Delta-like 1 serves as a new target and contributor to liver fibrosis down-regulated by mesenchymal stem cell transplantation [J]. J Biol Chem, 2011, 286(14): 12340-12348.
[10] Zhu NL, Asahina K, Wang J, et al. Hepatic stellate cell-derived delta-like homolog 1(DLK1) protein in liver regeneration [J]. J Biol Chem, 2012, 287(13): 10355-10367.
[11] Ohno N, Izawa A, Hattori M, et al. DLK inhibits stem cell factor-induced colony formation of murine hematopoietic progenitors: Hes-1- independent effect [J]. Stem Cells, 2001, 19(1): 71-79.
[12] Halder SK, Takemori H, Hatano O, et al. Cloning of a membrane-spanning protein with epidermal growth factor-like repeat motifs from adrenal glomerulosa cells[J]. Endocrinology, 1998, 139(7): 3316-3328.
[13] Floridon C, Jensen CH, Thorsen P, et al. Does fetal antigen 1 (FA1) identify cells with regenerative, endocrine and neuroendocrine potentials? A study of FA1 in embryonic, fetal, and placental tissue and in maternal circulation[J]. Differentiation, 2000, 66(1): 49-59.
[14] Harkness L, Taipaleenmaki H, Mahmood A, et al. Isolation and differentiation of chondrocytic cells derived from human embryonic stem cells using DLK1/FA1 as a novel surface marker [J].Stem Cell Rev, 2009, 5(4): 353-368.
[15] Sánchez-Solana B, Nueda ML, Ruvira MD, et al. The EGF-like proteins DLK1 and DLK2 function as inhibitory non-canonical ligands of NOTCH1 receptor that modulate each other's activities [J].Biochim Biophys Acta, 2011, 1813(6): 1153-1164.
[16] Kronenberg HM. Developmental regulation of the growth plate [J]. Nature, 2003, 423(6937): 332-336.
[17] Kronenberg HM. PTHrP and skeletal development[J]. Ann N Y Acad Sci, 2006, 1068: 1-13.
[18] Michigami T. Regulatory mechanisms for the development of growth plate cartilage [J]. Cell Mol Life Sci, 2013, 70(22): 4213-4221.
[19] Atsumi T, Miwa Y, Kimata K, et al. A chondrogenic cell line derived from a differentiating culture of AT805 teratocarcinoma cells [J]. Cell Differ Dev, 1990, 30(2): 109-116.
[20] Shukunami C, Ishizeki K, Atsumi T, et al. Cellular hypertrophy and calcification of embryonal carcinoma-derived chondrogenic cell line ATDC5 in vitro[J].J Bone Miner Res, 1997, 12(8): 1174-1188.