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大鼠骨髓间充质干细胞分化的内皮样细胞促进血管新生及Rho激酶的作用

郑惠珍^*, 赵静

广东医学院生理学研究室，生理学教研室，东莞 523808

摘要

本文研究大鼠骨髓间充质干细胞分化生成的内皮样细胞(rat bone marrow mesenchymal stem cells-differentiated endothelial like cells, rBMSC-ECs)在血管新生中的作用及Rho激酶(Rho kinase, ROCK)活性抑制的影响。实验建立rBMSC-ECs与主动脉环体外共培养实验模型，设单纯血管环组、血管环与细胞共培养组和HA-1077低、中、高浓度组，HA-1077组在共培养的基础上分别在培养液中加不同浓度(10、30、60 mmol/L)的ROCK特异抑制剂HA-1077。结果显示，培养第3天，血管环与细胞共培养组新生微血管数是单纯血管环组的1.3倍(P < 0.05)；HA-1077 10、30和60 mmol/L组较共培养组分别减少57.70%、64.13%和48.23% (均P < 0.01)。第6天，共培养组及HA-1077组rBMSC-ECs数量明显增加，并迁移至血管环周边，新生微血管生长缓慢；HA-1077组新生微血管数较共培养组明显减少。第9天，共培养组新生微血管部分增粗、增厚、延长，部分退化；一些rBMSC-ECs出芽，形成毛细血管样外观；而HA-1077组新生微血管几乎完全退化。Western blot检测血管环ROCKⅠ和ROCKⅡ的蛋白表达，与单纯血管环组比较，血管环与细胞共培养组表达稍增强，HA-1077各浓度组的表达稍降低。罗丹明-鬼笔毒环肽染色，激光共聚焦显微镜下观察显示，血管环新生微血管分布大量的肌动蛋白(F-actin)，细胞内呈现大量粗大的应力纤维，细胞前沿向前方伸出富含F-actin的丝状伪足。另一种ROCK特异抑制剂Y-27632 (10 μmol/L)抑制新生微血管细胞应力纤维形成，F-actin分布于细胞周边，细胞前沿丝状伪足消失。结果提示，rBMSC-ECs自身可分化为新生微血管，与动脉环体外共培养促进血管新生，ROCK抑制剂抑制其促血管新生作用，其机制涉及F-actin细胞骨架蛋白重组。

关键词： Rho激酶; 血管新生; 间充质干细胞; 主动脉环; 内皮分化; 骨髓; 大鼠

分类号：R329

[Rat bone marrow mesenchymal stem cells-differentiated endothelial like cells facilitate angiogenesis and the Rho kinase contribution.] [Article in Chinese]

ZHENG Hui-Zhen^*, ZHAO Jing

Department of Physiology, Guangdong Medical College, Dongguan 523808, China

Abstract

The aim of this study was to investigate the influence of endothelial like cells differentiated from rat bone marrow mesenchymal stem cells (rBMSC-ECs) on angiogenesis and the effect of Rho kinase (ROCK) inhibitor using an in vitro model of cells co-cultured with rat aorta ring. Cell proliferation capability was detected by MTT method. The rBMSC-ECs were co-cultured with rat aorta ring in rat tail collagen and endothelial medium. A ROCK specific inhibitor, HA-1077 at different concentrations (0, 10, 30 and 60 mmol/L, respectively) was added into the medium of ring-cell co-culture. The protein expression of ROCKⅠ and ROCKⅡ were detected by Western blot. On the third day of cultivation, the branch number of neogenetic microvessels increased by 34.5% in ring-cell co-culture group compared with that in simple aorta ring group (P < 0.01). Compared with that in ring-cell co-culture group, it was significantly decreased by 57.70%, 64.13% and 48.23% respectively in three concentrations of HA-1077 groups (all P < 0.01). However, on the sixth day, rBMSC-ECs proliferated and migrated to the nearby aorta ring, and the growth of microvessels became slow. On the ninth day, some of neogenetic microvessels were retracted, some became thicken, coarsen and lengthen, and some of rBMSC-ECs were sprouting and forming capillary like picture. The protein expression of ROCKⅠ/Ⅱ was slightly higher in ring-cell co-culture group than that in simple aorta ring group. But, in three concentrations of HA-1077 groups, it was slightly lower than that in ring-cell co-culture group. By using rhodamine-phalloidin staining and laser scanning confocal fluorescence microscope, it showed that there were a lot of the F-actin cytoskeletons in neogenesis microvessels of aorta ring, and there were a lot of thick and long stress fibers in the cells. F-actin-rich surface protrusions at the leading edge of the cell were also shown. Another ROCK inhibitor, Y-27632 (10 μmol/L) induced the actin cytoskeleton reorganization: F-actins appeared to be peripheral fibers at outer area of cell; stress fiber and filopodia disappeared. These results suggest that rBMSC-ECs themselves can be differentiated into new microvessels and facilitate angiogenesis when they are co-cultured with rat aorta ring. The mechanisms involve ROCK activation and F-actin cytoskeleton recombination.

Key words: Rho kinase; angiogenesis; mesenchymal stem cells; aorta ring; endothelial differentiation; bone marrow; rat

收稿日期：2011-02-23　　录用日期：2011-06-01

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

引用本文：

郑惠珍, 赵静. 大鼠骨髓间充质干细胞分化的内皮样细胞促进血管新生及Rho激酶的作用[J]. 生理学报 2011; 63 (4): 359-366.

ZHENG Hui-Zhen, ZHAO Jing. [Rat bone marrow mesenchymal stem cells-differentiated endothelial like cells facilitate angiogenesis and the Rho kinase contribution.] [Article in Chinese]. Acta Physiol Sin 2011; 63 (4): 359-366 (in Chinese with English abstract).