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法舒地尔促进大鼠骨髓间充质干细胞分化生成的内皮细胞迁移及血管新生

郑惠珍^*, 秋辉, 范爱辉

广东医学院生理学研究室，生理学教研室，湛江 524023

摘要

本文旨在研究Rho 激酶特异抑制剂法舒地尔(HA-1077)对大鼠骨髓间充质干细胞(rat bone marrow mesenchymal stemcells, rBMSCs)分化生成的内皮细胞的迁移和血管新生的影响，及其与血管内皮生长因子(vascular endothelial growth factor,VEGF)的关系。实验用第2~3 代rBMSCs，内皮细胞生长培养基EGM-2 诱导10~14 d，检测诱导后细胞的内皮标志。取诱导鉴定后的细胞，用跨膜趋化实验和划痕实验观察细胞的迁移率，用ECMatrixTM 凝胶体外血管新生实验观察血管新生情况。结果显示，HA-1077 能促进细胞迁移，其作用随浓度增高而增强，与VEGF 有明显的协同效应。细胞在ECMatrixTM凝胶上37 ºC 孵育12 h，细胞定向迁移，呈多边形排列，出芽，伸出的细长突起相互连接，形成封闭的多边形和复杂的毛细血管样网络结构。HA-1077 还能促进血管新生，新生毛细管总长度明显多于对照组(P<0.05)。在高浓度HA-1077 (60mmol/L)组中，细胞的主要表现为原位出芽，伸出细长突起，形成网络。当HA-1077 与VEGF 联合应用时，细胞的表现以形成复杂的毛细血管样网络结构为主。上述结果提示，HA-1077 能促进rBMSCs 分化生成的内皮细胞发生迁移和血管新生，并与VEGF 有一定程度的协同作用。

关键词： 骨髓; 间充质干细胞; 法舒地尔; 血管内皮生长因子; 血管新生; 细胞迁移; 大鼠

分类号：R329

[Fasudil promotes migration and vasculogenesis of endothelial cells differentiated from rat bone marrow mesenchymal stem cells in vitro.] [Ariticle in Chinese]

ZHENG Hui-Zhen^*, QIU Hui, FAN Ai-Hui

Laboratory of Physiology, Department of Physiology, Guangdong Medical College, Zhanjiang 524023, China

Abstract

The present study was designed to test whether Rho-kinase (ROCK) specific inhibitor fasudil (HA-1077) could contribute to migration and vasculogenesis of endothelial cells differentiated from rat bone marrow mesenchymal stem cells (rBMSCs) in vitro. rBMSCs were separated by gradient centrifugation on lymphocytes separation medium from bone marrow of Sprague-Dawley rats, and were cultured, purified and expanded in vitro. Cells of passage 2 to 3 were induced to differentiate into endothelial lineage cells by HG-DMEM plus EGM-2. These cells were identified as endothelial cells with positive factor VIII and Ulex europaeus agglutinin-1 expressions and DiI-Ac-LDL uptake. HA-1077 and VEGF synergistically promoted cell migration, especially in response to transwell chamber assay. When the cells were cultured on ECMatrixTM, they showed cellular protrusions and/or cords of aligned cells resembling primitive capillary-like structures at 8 to 12 h of incubation. HA-1077 promoted cell migration and formation of capillary-like tubes. The length of the total capillary tubes was longer than that in the control group (P<0.05). When the cells were exposed to a combination of VEGF and HA-1077, the number of the capillary-like networks and the stability of tube increased. The results obtained suggest that HA-1077 can promote migration and vasculogenesis of endothelial cells differentiated from rBMSCs in vitro.

Key words: Bone marrow; mesenchymal stem cell; fasudil; vascular endothelial growth factor; vasculogenesis; migration; rat

收稿日期：2009-02-17　　录用日期：2009-06-16

通讯作者：郑惠珍　　E-mail: zhenghz6@gdmc.edu.cn

引用本文：

郑惠珍, 秋辉, 范爱辉. 法舒地尔促进大鼠骨髓间充质干细胞分化生成的内皮细胞迁移及血管新生[J]. 生理学报 2009; 61 (4): 339-346.

ZHENG Hui-Zhen, QIU Hui, FAN Ai-Hui. [Fasudil promotes migration and vasculogenesis of endothelial cells differentiated from rat bone marrow mesenchymal stem cells in vitro.] [Ariticle in Chinese] . Acta Physiol Sin 2009; 61 (4): 339-346 (in Chinese with English abstract).