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

doi:10.19723/j.issn.1671-167X.2019.05.018

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

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. 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 WANG^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)

摘要/Abstract

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:

谢静,赵玉鸣,饶南荃,汪晓彤,方滕姣子,李晓霞,翟越,李静芝,葛立宏,王媛媛. 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[J]. 北京大学学报（医学版）, 2019, 51(5): 900-906.

Jing XIE,Yu-ming ZHAO,Nan-quan RAO,Xiao-tong WANG,Teng-jiao-zi FANG,Xiao-xia LI,Yue ZHAI,Jing-zhi LI,Li-hong GE,Yuan-yuan WANG. Comparative study of differentiation potential of mesenchymal stem cells derived from orofacial system into vascular endothelial cells[J]. Journal of Peking University(Health Sciences), 2019, 51(5): 900-906.

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参考文献 21

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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

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

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

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