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血管平滑肌细胞在不同细胞外基质的迁移特性: 玻片倒扣法研究

王凤, 王桂芹, 薛峰, 陈兆琴, 龚永生, 黄智慧^*

温州医学院低氧医学研究所，温州 325035

摘要

血管平滑肌细胞(vascular smooth muscle cells, VSMCs)迁移参与血管发育和多种疾病的发生、发展过程，然而其迁移机制尚不清楚。本课题建立了一种适合研究VSMCs迁移的方法——玻片倒扣法，基于该方法进一步研究VSMCs在不同细胞外基质上的迁移特性及机制。原代培养大鼠肺动脉平滑肌细胞(pulmonary arterial smooth muscle cells, PASMCs)，采用免疫细胞化学方法鉴定其纯度。将细胞接种于小玻片上，待生长至单层汇合后，将小玻片倒扣于包被有不同细胞外基质的大玻片上，培养24 h后，固定、拍照、统计软件测量迁移出的细胞数量和迁移距离。进一步采用细胞划痕实验和免疫细胞化学技术，研究细胞外基质对PASMCs迁移的作用及机制。结果显示：(1)原代培养PASMCs纯度高达(97 ± 3)%；(2)玻片倒扣24 h后，层粘连蛋白(laminin)组、基质胶(Matrigel)组比对照(phosphate buffered saline, PBS)组、多聚右旋赖氨酸(poly-D-lysine hydrobromide, PDL)组迁出的细胞数量更多，迁移距离更远，差异具有明显统计学意义；(3)细胞划痕8、12、24 h后，细胞在层粘连蛋白、基质胶上的面积愈合率比在PBS上的高，差异具有统计学意义；(4)免疫细胞化学结果显示迁移能力较强的PASMCs中肌动蛋白在与细胞运动方向相一致的边缘聚集现象越明显；vinculin (粘附斑的标记物)染色显示，种植于层粘连蛋白和基质胶上的PASMCs中，vinculin的分布较PBS和PDL上的更少。上述结果表明：玻片倒扣法是一种适合研究VSMCs迁移的方法；层粘连蛋白和基质胶可能通过抑制粘附斑的形成和调节骨架蛋白，促进VSMCs迁移。

关键词： 肺动脉平滑肌细胞; 细胞外基质; 迁移能力; 玻片倒扣

分类号：Q2-33

[Migratory properties of vascular smooth muscle cells on extracellular matrix: a study on inverted coverslip migration assay.] [Article in Chinese]

WANG Feng, WANG Gui-Qin, XUE Feng, CHEN Zhao-Qin, GONG Yong-Sheng, HUANG Zhi-Hui^*

Institute of Hypoxia Medicine, Wenzhou Medical College, Wenzhou 325035, China

Abstract

Migration of vascular smooth muscle cells (VSMCs) is involved in vascular development and various vascular diseases; however, the molecular mechanisms of VSMC migration remain unclear. In this study, we established an inverted coverslip migration assay to study the migratory properties of cultured VSMCs on extracellular matrix. Pulmonary arterial smooth muscle cells (PASMCs) from rats were cultured and identified by immunocytochemistry. Each coverslip with a confluent monolayer of PASMCs was inverted to a larger coverslip which was coated with phosphate buffered saline (PBS, as a control), poly-D-lysine hydrobromide (PDL), laminin or Matrigel. After 24 h of migration over the larger coverslip, PASMCs were fixed, and reliably quantified. The roles and mechanisms of extracellular matrix in PASMC migration were further studied by wound-healing assay and immunocytochemistry. The results showed that: (1) The purity of the cultured PASMCs was (97 ± 3)%. (2) The number of PASMCs on laminin or Matrigel migrating out from the inverted coverslip was significantly increased compared with that on PBS or PDL, and the migratory distance of PASMCs on laminin or Matrigel was significantly farther than that on PBS or PDL. (3) The motility of PASMCs on laminin or Matrigel was significantly higher than that on PBS at 8 h, 12 h and 24 h after wounding, respectively. (4) F-actin staining showed that F-actin was congregated along the brim of the migrating cells from the inverted coverslip, and vinculin (a cell marker of focal adhesion) staining showed that the distribution of vinculin in PASMCs plated on laminin or Matrigel was significantly lower than that on PBS or PDL. These results suggest that the inverted coverslip migration assay is suitable to study VSMC migration, and laminin and Matrigel substrates may promote VSMC migration through inhibiting the formation of focal adhesion and regulating the cytoskeletal proteins.

Key words: pulmonary arterial smooth muscle cells; extracellular matrix; motility; inverted coverslip

收稿日期：2012-07-09　　录用日期：2012-12-14

通讯作者：黄智慧　　E-mail: hzhzju021@163.com

引用本文：

王凤, 王桂芹, 薛峰, 陈兆琴, 龚永生, 黄智慧. 血管平滑肌细胞在不同细胞外基质的迁移特性: 玻片倒扣法研究[J]. 生理学报 2013; 65 (2): 135-142.

WANG Feng, WANG Gui-Qin, XUE Feng, CHEN Zhao-Qin, GONG Yong-Sheng, HUANG Zhi-Hui. [Migratory properties of vascular smooth muscle cells on extracellular matrix: a study on inverted coverslip migration assay.] [Article in Chinese]. Acta Physiol Sin 2013; 65 (2): 135-142 (in Chinese with English abstract).