基因转染EphB4对脱落乳牙牙髓干细胞成骨分化的影响

doi:10.19439/j.sjos.2017.04.010

上海口腔医学 ›› 2017, Vol. 26 ›› Issue (4): 399-403.doi: 10.19439/j.sjos.2017.04.010

基因转染EphB4对脱落乳牙牙髓干细胞成骨分化的影响

李晓明^{1, 2}, 袁长永², 朱绍跃², 刘宗响², 王鹏来²

1.徐州医科大学口腔医学院,江苏徐州 221004；
2.徐州医科大学附属徐州口腔医院,江苏徐州 221002

收稿日期:2016-12-19 修回日期:2017-03-20 出版日期:2017-08-25 发布日期:2017-09-01
通讯作者: 王鹏来,E-mail: wpl0771@163.com
作者简介:李晓明（1990-）,女,硕士,E-mail:shuiming99@126.com
基金资助:
徐州市科技计划项目（Z201628）

Effect of transfected EphB4 on osteogenic differentiation in stem cells from human exfoliated deciduous teeth

LI Xiao-ming^{1, 2}, YUAN Chang-yong², ZHU Shao-yue², LIU Zong-xiang², WANG Peng-lai²

1.School of Stomatology, Xuzhou Medical University. Xuzhou 221004;
2.Xuzhou Stomatological Hospital Affiliated to Xuzhou Medical University. Xuzhou 221002, Jiangsu Province, China

Received:2016-12-19 Revised:2017-03-20 Online:2017-08-25 Published:2017-09-01

摘要/Abstract

摘要： 目的建立过表达EphB4的脱落乳牙牙髓干细胞（stem cells from human exfoliated deciduous teeth,SHED）,观察其成骨分化能力的变化。方法使用酶消化法分离、培养SHED,将第3代SHED分为2组,即病毒介导的EphB4基因转染SHED的实验组和空白荧光载体转染SHED的对照组。通过荧光显微镜、实时荧光定量PCR（real time-PCR）检测转染后SHED细胞中EphB4 mRNA的表达水平,Western 印迹法检测转染后EphB4蛋白的表达量。成骨诱导4周后,使用碱性磷酸酶（alkaline phosphatase,ALP）试剂盒及茜素红染色测定转染后SHED的ALP活性改变和成骨能力变化。采用SPSS16.0软件包对数据进行统计学分析。结果实时荧光定量PCR显示,实验组细胞中EphB4基因表达增高约8倍（P<0.05）,Western印迹法显示,实验组细胞中EphB4蛋白表达较对照组显著增高。成骨诱导4周后,ALP检测结果表明,基因转染EphB4,可显著促进SHED的ALP活性（P<0.05）,增高约2倍;茜素红染色显示,转染EphB4后,SHED成骨能力显著增强。结论基因转染EphB4可显著促进SHED的成骨分化能力,为SHED成为骨组织工程中的种子细胞提供了依据。

关键词: 脱落乳牙牙髓干细胞, 基因转染, EphB4/ephrinB2, 成骨诱导, 细胞分化

Abstract: To evaluate the effect of stem cells from human exfoliated deciduous teeth (SHED) transfected with EphB4 gene in regulating osteogenic differentiation. METHODS: Human dental pulp tissue were harvested from extracted deciduous teeth and digested by collagenase and dispase. The SHEDs were transfected with transgenic (hEphB4-GFP) vector or empty vector (GFP-vector). Real time-polymerase chain reaction（real time-PCR） analysis and Western blot were used to detect the expression of EphB4 in SHEDs after transfection. EphB4-SHEDs and GFP-SHEDs were subjected to osteogenic induction and assessed by alkaline phosphatase(ALP) assay and Alizarin-red S staining. SPSS 16.0 software package was used for statistical analysis. RESULTS: Real time-PCR revealed that the expression of EphB4 was significantly enhanced in EphB4-SHEDs compared to GFP-SHEDs (P<0.05). The expression of EphB4 protein was significantly higher (P<0.05) in EphB4-SHEDs compared to GFP-SHEDs. ALP assay and Alizarin-red S staining demonstrated higher ALP activity and increased mineralization with EphB4-SHEDs. CONCLUSIONS: The results indicate that transgenic expression of EphB4 in SHEDs could promote osteogenic differentiation.

Key words: SHED, Gene transfection, EphB4/ephrinB2, Osteogenic induction, Cell differentiation

中图分类号:

R781.3

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.

LI Xiao-ming, YUAN Chang-yong, ZHU Shao-yue, LIU Zong-xiang, WANG Peng-lai. Effect of transfected EphB4 on osteogenic differentiation in stem cells from human exfoliated deciduous teeth[J]. Shanghai Journal of Stomatology, 2017, 26(4): 399-403.

参考文献

[1] Amini AR, Laurencin CT, Nukavarapu SP. Bone tissue engineering: recent advances and challenges [J]. Crit Rev Biomed Eng, 2012, 40(5): 363-408.
[2] O'Keefe RJ, Mao J. Bone tissue engineering and regeneration: from discovery to the clinic-an overview [J]. Tissue Eng Part B Rev, 2011, 17(6): 389-392.
[3] Langer R, Vacanti JP. Tissue engineering [J]. Science, 1993, 260(5110): 920-926.
[4] Jakobsen C, S?尴rensen JA, Kassem M, et al. Mesenchymal stem cells in oral reconstructive surgery: a systematic review of the literature [J]. J Oral Rehabil, 2013, 40(9): 693-706.
[5] Chadipiralla K, Yochim JM, Bahuleyan B, et al. Osteogenic differentiation of stem cells derived from human periodontal ligaments and pulp of human exfoliated deciduous teeth[J]. Cell Tissue Res, 2010, 340(2): 323-333.
[6] ZigdonGiladi H, Khoury N, Evron A. Adult stem cells in the use of jaw bone regeneration: current and prospective research [J]. Quintessence Int, 2015, 46(2): 125-131.
[7] MacHado E, Fernandes MH, SousaGomes P. Dental stem cells for craniofacial tissue engineering [J]. Oral Surg Oral Med Oral Pathol Oral Radiol, 2012, 113(6): 728-733.
[8] Edwards CM, Mundy GR. Eph receptors and ephrin signalling pathways: a role in bone homeostasis[J]. Int J Med Sci, 2008,5(5): 263-272.
[9] Miura M, Gronthos S, Zhao M, et al. SHED: stem cells from human exfoliated deciduous teeth [J]. Proc Natl Acad Sci USA, 2003, 100(10): 5807-5812.
[10] Farea M, Husein A, Halim AS, et al. Synergistic effects of chitosan scaffold and TGFβ1 on the proliferation and osteogenic differentiation of dental pulp stem cells derived from human exfoliated deciduous teeth [J]. Arch Oral Biol, 2014, 59(12): 1400-1411.
[11] Wang X, Sha XJ, Li GH, et al. Comparative characterization of stem cells from human exfoliated deciduous teeth and dental pulp stem cells [J]. Arch Oral Biol, 2012, 57(9): 1231-1240.
[12] Suchánek J, Visek B, Soukup T, et al. Stem cells from human exfoliated deciduous teeth-isolation, long term cultivation and phenotypical analysis [J]. Acta Medica (Hradec Kralove), 2010, 53(2): 93-99.
[13] Behnia A, Haghighat A, Talebi A, et al. Transplantation of stem cells from human exfoliated deciduous teeth for bone regeneration in the dog mandibular defect [J]. World J Stem Cells, 2014, 6(4): 505-510.
[14] Mundy GR, Elefteriou F. Boning up on ephrin signaling [J]. Cell, 2006, 126(3): 441-443.
[15] Zhao C, Irie N, Takada Y, et al. Bidirectional ephrinB2-EphB4 signaling controls bone homeostasis [J]. Cell Metab, 2006, 4(2): 111-121．
[16] Takyar FM, Tonna S, Ho PW, et al. EphrinB2/EphB4 inhibition in the osteoblast lineage modifies the anabolic response to parathyroid hormone [J]. J Bone Miner Res, 2013, 28(4): 912-925.
[17] Tonna S, Takyar FM, Vrahnas C, et al.EphrinB2 signaling in osteoblasts promotes bone mineralization by preventing apoptosis [J]. FASEB J, 2014, 28(10): 4482-4496.
[18] Ma X, Luo D, Li K, et al. Suppression of EphB4 improves the inhibitory effect of mTOR shRNA on the biological behaviors of ovarian cancer cells by down-regulating akt phosphorylation [J]. J Huazhong Univ Sci Technolog Med Sci, 2012, 32(3): 358-563.
[19] Pasquale EB. Eph-ephrin bidirectional signaling in physiology and disease [J]. Cell, 2008, 133(1): 38-52.
[20] Li C, Shi C, Kim J, et al. Erythropoietin promotes bone formation through EphB4/EphrinB2 signaling [J]. J Dent Res, 2015, 94(3): 455-463.
[21] Tierney EG, McSorley K, Hastings CL, et al. High levels of ephrinB2 over-expression increases the osteogenic differentiation of human mesenchymal stem cells and promotes enhanced cell mediated mineralisation in a polyethyleneimine-ephrinB2 gene-activated matrix [J]. J Control Release, 2013, 165(3): 173-182.
[22] Zhu SY, Wang PL, Liao CS, et al. Transgenic expression of ephrinB2 in periodontal ligament stem cells (PDLSCs) modulates osteogenic differentiation via signaling crosstalk between ephrinB2 and EphB4 in PDLSCs and between PDLSCs and pre-osteoblasts within co-culture[J].J Periodontal Res, 2017,52(3):562-573.

基因转染EphB4对脱落乳牙牙髓干细胞成骨分化的影响

Effect of transfected EphB4 on osteogenic differentiation in stem cells from human exfoliated deciduous teeth

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