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北京大学学报（医学版） ›› 2019, Vol. 51 ›› Issue (5): 900-906. doi: 10.19723/j.issn.1671-167X.2019.05.018

• 论著 • 上一篇    下一篇

3种口腔颌面部来源的间充质干细胞成血管内皮分化潜能的比较研究

谢静1,赵玉鸣1,饶南荃1,汪晓彤2,方滕姣子1,李晓霞1,翟越1,李静芝1,葛立宏1,王媛媛1,△()   

  1. 1. 北京大学口腔医学院·口腔医院,儿童口腔科　国家口腔疾病临床医学研究中心　口腔数字化医疗技术和材料国家工程实验室　口腔数字医学北京市重点实验室,北京　100081
    2. 北京大学口腔医学院·口腔医院,急诊科　国家口腔疾病临床医学研究中心　口腔数字化医疗技术和材料国家工程实验室　口腔数字医学北京市重点实验室,北京　100081
  • 收稿日期:2017-10-10 出版日期:2019-10-18 发布日期:2019-10-24
  • 通讯作者: 王媛媛 E-mail:cwyyd@126.com
  • 基金资助:
    国家自然科学基金(81500837)

Comparative study of differentiation potential of mesenchymal stem cells derived from orofacial system into vascular endothelial cells

Jing XIE1,Yu-ming ZHAO1,Nan-quan RAO1,Xiao-tong WANG2,Teng-jiao-zi FANG1,Xiao-xia LI1,Yue ZHAI1,Jing-zhi LI1,Li-hong GE1,Yuan-yuan WANG1,△()   

  1. 1. Department of Pediatric Dentistry, Peking University School and Hospital of Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Laboratory for Digital and Material Technology of Stomatology & Beijing Key Laboratory of Digital Stomatology, Beijing 100081, China
    2. Department of Oral Emergency, Peking University School and Hospital of Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Laboratory for Digital and Material Technology of Stomatology & Beijing Key Laboratory of Digital Stomatology, Beijing 100081, China
  • Received:2017-10-10 Online:2019-10-18 Published:2019-10-24
  • Contact: Yuan-yuan WANG E-mail:cwyyd@126.com
  • Supported by:
    Supported by National Natural Science Youth Foundation of China(81500837)

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摘要：

目的:研究来自口腔颌面部的脱落乳牙牙髓干细胞(stem cells from human exfoliated deciduous teeth, SHED)、牙髓干细胞(dental pulp stem cells, DPSC)和颌骨骨髓间充质干细胞(bone marrow mesenchymal stem cell, BMSC)的增殖能力及成血管内皮细胞分化潜能,为血管组织工程再生种子细胞的选择提供依据。方法:取临床乳牙和恒牙牙髓组织及颌骨组织并采用酶消化法分离培养相应的间充质干细胞,流式细胞技术检测间充质干细胞相关表面抗原的表达。采用CCK-8 (cell counting kit-8)法检测细胞的增殖能力。通过Matrigel三维培养技术诱导间充质干细胞成血管内皮细胞分化,并通过管腔计数及real-time PCR技术比较3种间充质干细胞的成血管内皮细胞分化能力。采用鸡胚绒毛尿囊膜(chick embryo chorioallantoic membrane, CAM)技术观察3种不同间充质干细胞新生血管能力。结果:3种干细胞均阳性表达CD73、CD90、CD105、CD146,阴性表达 CD34、CD45,符合间充质干细胞表面标记物的表达规律。SHED和DPSC的CD146表达率多于BMSC,CCK-8法检测显示SHED的增殖能力最强。诱导后的3种细胞均可在Matrigel基质胶上形成管腔样结构,诱导后SHED和BMSC形成的血管总长度大于DPSC,SHED形成的管腔数多于BMSC和DPSC。real-time PCR 结果显示几种成血管相关的细胞因子在不同细胞间表达存在差别。诱导后SHED的CD31、VEGFR2、vWF表达显著高于另外两种细胞。BMSC的VEGFR1表达量高于其他组,SHED高于DPSC。VEGF的表达在4组之间差异无统计学意义。3种细胞在CAM上计数新生血管数目显示较空白对照组差异无统计学意义,SHED组血管总长度较空白对照和BMSC大。结论:SHED、DPSC及 BMSC均能向成血管内皮细胞方向诱导分化且在Matrigel培养基上形成血管和管腔,SHED具有更强的分化和形成管腔的能力,同时SHED较BMSC及DPSC具有更强的增殖能力。

关键词: 脱落乳牙牙髓干细胞, 牙髓干细胞, 颌骨骨髓间充质干细胞, 血管内皮细胞分化, 细胞增殖

Abstract:

Objective: To compare the proliferation and capacity of differentiation to vascular endothelial cells and angiogenesis induction among stem cells from human exfoliated deciduous teeth (SHED), dental pulp stem cells (DPSC) and human bone marrow mesenchymal stem cells (BMSC) from orofacial bone. Methods: SHED and DPSC were isolated from pulp tissue of the patients. BMSC were isolated from orthognathic or alveolar surgical sites. The surface markers of the cells were detected by flowcytometry. Cell counting kit-8 (CCK-8) assays were conducted to detect the proliferation ability of the cells. The cells were induced into endothelial cells with conditional medium and then the induced cells were cultured in Matrigel medium. The expression of angiogenesis-related genes such as platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31), vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor 1 (VEGFR1), vascular endothelial growth factor receptor 2 (VEGFR2) and von Willebrand Factor (vWF) were quantified by real-time PCR. The cells were cultured in chick embryo chorioallantoic membrane (CAM) and the vessels were counted after 5 days. Results: The cell surface markers CD73, CD90, CD105 and CD146 of all the stem cells were positive, CD34 and CD45 were negative. The CD146 positive rate of SHED and DPSC was higher than that of BMSC. SHED had a higher proliferation rate than DPSC and BMSC. After angiogenic induction for 14 d, 3 kinds of cells emanated pseudopodia formed grid structure long vasculature in Matrigel media. The total length of tube formation of induced BMSC (7 759.7 μm) and SHED (7 734.3 μm) was higher than DPSC (5 541.0 μm). The meshes number of induced SHED (70.7) was higher than DPSC (60) and BMSC (53.7) in Matrigel medium. The expression of CD31, VEGFR2 and vWF genes of SHED were higher than those of BMSC and DPSC. VEGFR1 gene expression of BMSC was higher than that of the other groups, and SHED was higher than DPSC. The expression of VEGF showed no difference among the cells. No deference was showed between the effect of the stem cells and negative control on new formed vessels in CAM. The total length of vessels of SHED (30.4 mm) was higher than that of the negative control (20.9 mm) and BMSC (28.0 mm). Conclusion: SHED, DPSC and BMSC can differentiate into vascular endothelial cells. SHED showed a stronger angiogenesis differentiation and proliferation potential compared with DPSC and BMSC.

Key words: Stem cells from human exfoliated deciduous teeth, Dental pulp stem cells, Bone marrow mesenchymal stem cells, Differentiation into vascular endothelial cells, Cell proliferation

中图分类号: 

  • R78

表1

基因及real-time PCR引物序列"

Gene Primer(5' to 3') Size/bp
CD31 F: TGTCAAGTAAGGTGGTGGAGTCT
R: AGGCGTGGTTGGCTCTGTT		 222
VEGF F: CCCACTGAGGAGTCCAACAT
R: AAATGCTTTCTCCGCTCTGA		 296
VEGFR1 F: ACCTCACTGCCACTCTAATTGTCA
R: AAGTCACACCTTGCTTCGGAATG		 196
VEGFR2 F: ACGGACAGTGGTATGGTTCTTGCC
R: GGTAGCCGCTTGTCTGGTTTGAG		 145
vWF F: GGGGTCATCTCTGGATTCAAG
R: TCTGTCCTCCTCTTAGCTGAA		 145
GAPDH F: CTGGGCTACACTGAGCACC
R: AAGTGGTCGTTGAGGGCAATG		 101

图1

流式细胞仪检测细胞表面标志结果"

图2

BMSC、DPSC和SHED增殖能力的比较"

图3

诱导后的BMSC、DPSC和SHED在Matrigel三维培养基上形成管腔情况"

图4

诱导后的3种细胞血管内皮细胞相关基因的表达"

图5

BMSC、DPSC和SHED作用下CAM上形成的新生血管"

[1] Sakaguchi Y, Sekiya I, Yagishita K , et al. Comparison of human stem cells derived from various mesenchymal tissues: superiority of synovium as a cell source[J]. Arthritis Rheumatol, 2005,52(8):2521.
[2] Folkman J . Angiogenesis in cancer, vascular, rheumatoid and other disease[J]. Nat Med, 1995,1(1):27.
[3] Carmeliet P . Angiogenesis in life, disease and medicine[J]. Nature, 2005,438(7070):932.
[4] Canan A, Huseyin A, Riza KA , et al. Angiogenesis in inflammatory bowel disease[J]. In J Inflammation, 2015,2015(3):970890.
[5] Tateishi-Yuyama E, Matsubara H, Murohara T . Therapeutic angiogenesis for patients with limb ischemia by autologous transplantation of bone-marrow cells: a pilot study and a randomized controlled trial[J]. Acc Current J Rev, 2002,360(9331):427.
[6] Hou L, Kim JJ, Woo YJ , et al. Stem cell-based therapies to promote angiogenesis in ischemic cardiovascular disease[J]. Am J Physiol, 2016,310(4):H455.
[7] Shi S, Gronthos S . Perivascular niche of postnatal mesenchymal stem cells in human bone marrow and dental pulp[J]. J Bone Miner Res, 2003,18(4):696-704.
[8] Belotti A, Elli E, Speranza T , et al. Circulating endothelial cells and endothelial activation in essential thrombocythemia: results from CD146 + immunomagnetic enrichment: flow cytometry and soluble E-selectin detection [J]. Am J Hematol, 2012,87(3):319.
[9] Jouve N, Despoix N, Espeli M , et al. The involvement of CD146 and its novel ligand galectin-1 in apoptotic regulation of endothelial cells[J]. J Biol Chem, 2013,288(4):2571-2579.
[10] Nakamura S, Yamada Y, Katagiri W , et al. Stem cell proliferation pathways comparison between human exfoliated deciduous teeth and dental pulp stem cells by gene expression profile from promising dental pulp[J]. J Endodont, 2009,35(11):1536-1542.
[11] Duttenhoefer F, Lara d FR, Meury T, et al. 3D scaffolds co-seeded with human endothelial progenitor and mesenchymal stem cells: evidence of prevascularisation within 7 days[J]. Eur Cells Mater, 2013,26(4):49.
[12] Moore MC, Pandolfi V, Mcfetridge PS . Novel human-derived extracellular matrix induces in vitro, and in vivo, vascularization and inhibits fibrosis[J]. Biomaterials, 2015,49:37.
[13] Nourse MB, Halpin DE, Scatena M , et al. VEGF induces differentiation of functional endothelium from human embryonic stem cells: implications for tissue engineering[J]. Arterioscler Thromb Vas Biol, 2010,30(1):80-89.
[14] Oswald J, Boxberger S, Jørgensen B , et al. Mesenchymal stem cells can be differentiated into endothelial cells in vitro[J]. Stem Cells, 2004,22(3):377.
[15] Sugiyama M, Iohara K, Wakita H , et al. Dental pulp-derived CD31 -/CD146 - side population stem/progenitor cells enhance recovery of focal cerebral ischemia in rats [J]. Tissue Eng Part A, 2011,17(9/10):1303-1311.
[16] Park S, Sorenson CM, Sheibani N . PECAM-1 isoforms, eNOS and endoglin axis in regulation of angiogenesis[J]. Clin Sci, 2015,129(3):217.
[17] Zhang Z, Neiva KG, Lingen MW , et al. VEGF-dependent tumor angiogenesis requires inverse and reciprocal regulation of VEGFR1 and VEGFR2[J]. Cell Death Differ, 2010,17(3):499.
[18] Ruszkowskaciastek B, Sokup A, Socha M W , et al. A preliminary evaluation of VEGF-A, VEGFR1 and VEGFR2 in patients with well-controlled type 2 diabetes mellitus[J]. J Zhejiang Univ Sci B, 2014,15(6):575-581.
[19] Rondaij MG, Bierings R, Kragt A , et al. Dynamics and plasticity of Weibel-Palade bodies in endothelial cells[J]. Arterioscler Thromb Vasc Biol, 2006,26(5):1002.
[20] Kleibeuker EA, Schulkens IA, Castricum KC , et al. Examination of the role of galectins during in vivo angiogenesis using the chick chorioallantoic membrane assay[J]. Methods Mol Biol, 2015,1207:305.
[21] Dehelean CA, Feflea S, Gheorgheosu D , et al. Anti-angiogenic and anti-cancer evaluation of betulin nanoemulsion in chicken chorioallantoic membrane and skin carcinoma in BALB/c mice[J]. J Biomed Nanotechnol, 2013,9(4):577-589.
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ISSN 1671-167X
CN 11-4691/R
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