研究背景:
    在血管再狭窄形成过程中,血管中膜平滑肌细胞(Vascular smooth muscle cell, VSMC)向内膜下迁移并在内膜中增生是关键步骤.与细胞运动直接相关的肌动蛋白细胞骨架参与了VSMC迁移过程,从而促进再狭窄的发生.已知与肌动蛋白相关的细胞结构主要有:丝状伪足(Filopodia)、板状伪足(Lamellipodia)、粘着斑(Focal adhension)和张力纤维(Stress fiber).近年来的研究表明,Rho家族小G蛋白是细胞骨架肌动蛋白的重要调节因子,目前众多的研究集中在Rho小G蛋白家族成员中的Cdc42、Rac1和RhoA,其中Cdc42促进丝状伪足的形成,Rac1调节板装伪足的生成和膜皱缩,RhoA则促进粘着斑连接和张力纤维的装配.我们考虑在不同细胞类型以及不同活性物质作用下,小G蛋白信号转导途径可不同.因此,研究小G蛋白介导的人血管平滑肌细胞的细胞骨架构建的变化,有助于揭示血管再狭窄过程中VSMC迁移的信号转导机制.
    第一部分 IL-1α、PDGF-BB及LPA诱导的细胞骨架的构建目的:
    研究激活Rho家族小G蛋白的不同细胞因子所诱导的细胞骨架构建变化.
方法:
    本研究用激活Rho家族小G蛋白的细胞因子白细胞介素1α(IL-1α)、血小板源性生长因子(PDGF-BB)及溶血磷脂酸(LPA)诱导原代培养的人血管平滑肌细胞,免疫荧光标记与运动有关的肌动蛋白细胞骨架结构,激光共聚焦显微镜在不同时间点观察人VSMC细胞骨架构建变化情况.
结果:
    IL-1α诱导10分钟后出现丝装伪足,20分钟时出现板装伪足,30分钟时出现张力纤维和明显的粘着斑.PDGF-BB诱导5分钟后出现板装伪足,15分钟时出现张力纤维及粘着斑,30分钟时较前无明显改变.LPA诱导20分钟时出现张力纤维及粘着斑.
结论:
    IL-1α、PDGF-BB及LPA诱导后VSMC细胞骨架构建出现规律性的变化,提示Rho家族小G蛋白存在级联瀑布式的反应顺序.
Background:
    Intimal hyperplasia is the principal cause of restenosis of blood vessels following vascular intervention or injury. Migration from the media to intima and subsequent proliferation of vascular smooth muscle cells(VSMC) are key events in the early development of the intimal hyperplastic lesion. Thus the actin-based structures which have tight relation with the migration of VSMC take part in the development of restenosis. These structures include filopodia, lamellipodia, stress fiber and focal adhesion. Recent studies showed that Rho GTPase played a key role in the reorganization of cytoskeleton and in Swiss 3T3 cells, Cdc42, Rac1 and RhoA, three members of Rho superfamily of small GTPase, regulated the polymerization of actin to produce filopodia , lamellipodia, stress fiber and focal adhension, respectively. However, different cell types may undergo different signaling pathways. So the study of cytoskeleton change in human VSMCs induced by Rho GTPase is essential to clarify the signaling mechanism of migration of human VSMCs in vascular restenosis.
    Part Ⅰ Reorganization of cytoskeleton in human vascular smooth muscle cell after induction of L-1α, PDGF-BB and LPAObjectives:
    To study the reorganization of cytoskeleton in human VSMCs induced by Rho GTPase-activating cytokines.
Methods:
    Firstly, the primary human vascular smooth muscle cells were cultured. After inducement Rho GTPase-activating cytokines including IL-1α, PDGF-BB and LPA, F-actin was stained by FITC-labeled Phalloidine to visualize actin-based structure including filopodia, lamellipodia and stress fiber. And vinculin distribution of focal adhesion were visualized through indirect immunofluorescence. These cytoskeletons of VSMC were then observed with confocal laser scanning microscope at different time points.
Results:
    The filopodia, lamelloipodia, stress fibers and focal adhensions in VSMC appeared at 10min, 20min and 30min after inducement of IL-1α. respectively. PDGF-BB induced the production lamellipoddia, stress fibers at 5min and 15min respectively. And focal adhensions accompanied with stress fiber was observed 20min after inducement of LPA.
Conclusions:
    Rho GTPase-activating cytokines induced the reorganization of cytoskeleton in human VSMCs, which indicated inner activating sequence among members of Rho superfamily of small GTPase.

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