牵张力促进BMSCs与VECs共培养体系成骨分化的体外研究

上海口腔医学 ›› 2015, Vol. 24 ›› Issue (5): 400-404.

牵张力促进BMSCs与VECs共培养体系成骨分化的体外研究

王宇, 唐国华

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

收稿日期:2015-01-05 修回日期:2015-03-30 出版日期:2015-10-20 发布日期:2015-11-03
通讯作者: 唐国华,E-mail:drtanggh@gmail.com
作者简介:王宇(1988-),女,在读硕士研究生,E-mail:yuxiaoha@hotmail.com
基金资助:
国家自然科学基金(81271113); 上海市科学技术委员会科研基金(15ZR1425200)

Cyclic mechanical strain induces osteogenic differentiation in the coculture system of BMSCs and VECs

WANG Yu, TANG Guo-hua

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

Received:2015-01-05 Revised:2015-03-30 Online:2015-10-20 Published:2015-11-03
Supported by:
; Supported by National Natural Science Foundation of China (81271113) and Research Fund of Science and Technology Committee of Shanghai Municipality (15ZR1425200)

摘要/Abstract

摘要： 目的：研究牵张力对骨髓基质细胞（BMSCs）与血管内皮细胞（VECs）共培养体系成骨分化的作用及相关机制。方法分离培养大鼠原代BMSCs与VECs。应用Flexcell 5000加力系统,分别对BMSCs与VECs共培养组、BMSCs单独培养组和VECs单独培养组施加6%等轴循环牵张力。加力6、12、24和48 h后,利用实时定量PCR检测Runx2和血管内皮细胞生长因子(VEGF)mRNA的表达量,ELISA法检测细胞培养上清液中VEGF的含量,碱性磷酸酶（ALP）半定量检测ALP活性。通过加入VEGF受体抑制剂Tivozanib,观察VEGF的旁分泌作用。采用SAS 8.0软件包对数据进行统计学分析。结果①加力6 h时,共培养体系Runx2 mRNA的表达量上调4.3倍(P<0.05);加力48 h时,ALP活性升高1.5倍(P<0.05)。②加力12 h时,共培养体系VEGF mRNA的表达量上调2倍(P<0.05),上清液中VEGF的含量增加10倍(P<0.05),BMSCs分泌大量VEGF,而VECs分泌极少量VEGF。③加入Tivozanib后,共培养组Runx2的表达量下调90%(P<0.05),ALP活性下调48%(P<0.05);而 BMSCs单独培养组Runx2的表达量和ALP活性分别下降30%和18%。结论牵张力促进BMSCs与VECs共培养体系中BMSCs的成骨分化,这种作用可能通过牵张应力诱导BMSCs分泌的VEGF以旁分泌方式由VECs作用于BMSCs来实现。

关键词: 骨髓基质细胞, 血管内皮细胞, 共培养, 牵张应力, 成骨分化, VEGF

Abstract: PURPOSE:To detect the effects of cyclic mechanical strain on the coculture system of bone marrow stromal cells(BMSCs)and vascular endothelial cells(VECs)and to clarify the related mechanism. METHODS: Primary BMSCs and VECs were isolated from Sprague-Dawley rats. BMSCs and VECs coculture group, BMSCs monoculture group and VECs monoculture group were exposed to cyclic mechanical strain(6%) by Flexcell 5000 mechanicl loading system. After mechanical loading for 6, 12, 24 and 48h, the mRNA expression of Runx2 and vascular endothelial growth factor(VEGF)were tested by real-time quantitative PCR. VEGF protein in cell culture supernate was quantified using Elisa Kit and alkaline phosphatase(ALP)activity was examined. The VEGFR inhibitor Tivozanib was used to analyze the paracrine role of VEGF. Statistical analysis was performed using SAS 8.0 software package. RESULTS: ①The mRNA expression of Runx2 of coculture group subjected to 6% strain was increased significantly at 6h with 4.3 folds (P<0.05) and ALP activity at 48h with 1.5 folds(P<0.05). ②The mRNA expression of VEGF of coculture group subjected to 6% stain was increased significantly at 12h with 2 folds (P<0.05) and the content of VEGF in cell culture supernate at 12h with 10 folds (P<0.05). Plenty of VEGF was secreted by BMSCs but little by VECs. ③After being subjected to Tivozanib, the expression of Runx2 VECs. ③After being subjected to Tivozanib, the expression of Runx2 of coculture group was decreased by 90% (P<0.05) and ALP activity was decreased by 48% (P<0.05), while those of BMSCs monoculture group were decreased by 30% and 18%, respectively. CONCLUSIONS: Cyclic mechanical strain promotes osteogenic differentiation of BMSCs in the coculture system of BMSCs and VECs, possibly by a paracrine VECs-mediated effect of VEGF on BMSCs.

Key words: BMSCs, VECs, Coculture, Cyclic mechanical strain, Osteogenic differentiation, VEGF

王宇, 唐国华. 牵张力促进BMSCs与VECs共培养体系成骨分化的体外研究[J]. 上海口腔医学, 2015, 24(5): 400-404.

WANG Yu, TANG Guo-hua. Cyclic mechanical strain induces osteogenic differentiation in the coculture system of BMSCs and VECs[J]. Shanghai Journal of Stomatology, 2015, 24(5): 400-404.

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