【作者】 刘智敏；

【导师】 韩梅； 温进坤；

【作者基本信息】 河北医科大学 ， 生物化学与分子生物学， 2003， 博士

【摘要】 血管平滑肌细胞（vascular smooth muscle cell, VSMC）由中膜向内膜下迁移是血管内皮受损后发生的一种早期反应，普遍存在于动脉粥样硬化、经皮腔内冠脉成形术（percutaneous transluminal coronary angioplasty，PTCA）后再狭窄的病变中，是新生内膜形成的生物学基础。多种生长因子、炎性因子和细胞外基质 (extracellular matix, ECM) 均具有诱导VSMC迁移的作用。但是有关这些分子调节VSMC迁移的细胞内信号途径尚不十分清楚。近年来的研究发现，整合素作为ECM蛋白的受体不仅介导细胞与ECM、细胞与细胞之间的相互作用，而且还具有整合细胞外刺激信号向胞内转导的功能。粘着斑激酶（focal adhesion kinase，FAK）则是整合素信号途径中连接整合素与下级信号分子的媒介，是胞内多条信号途径的交汇点。FAK可借助其分子内含有的多个酪氨酸磷酸化位点催化与其结合的PI-3K等一系列信号分子的活化，进而触发级联磷酸化反应，将来自整合素的信号向下游传递，该信号途径在细胞收缩、变形及运动过程中具有重要的作用。我室多年来的研究结果表明，体外培养的或体内血管病变中存在的VSMC可合成分泌大量的ECM蛋白。其中，骨桥蛋白（osteopontin, OPN）作为合成型VSMC的标志物，普遍存在于体外培养的VSMC培养液中或体内血管病变中，且含量丰富。已经证实，OPN具有促进VSMC粘附的作用，VSMC通过细胞膜表面的整合素β3或β5 与OPN的相互作用，而使细胞得以固定在基质中。但是有关OPN对VSMC迁移的影响及可能的信号途径的研究报道甚少。虽然人们已经对整合素激活所触发的信号转导及其在细胞迁移中的意义进行了一系列的研究，但是整合素β3-FAK是否参与了OPN诱导的VSMC迁移过程尚缺乏直接的证据。本研究以OPN作为诱导因素，首先观察其对VSMC迁移的影响及其作用规律，探讨OPN介导的VSMC迁移与整合素β3-FAK信号途径活化之间的关系。在此基础上，<WP=5>通过转染外源性FRNK表达载体，以阻断FAK活性，以期进一步证实该信号途径在OPN诱导的VSMC迁移中的意义。试图从分子、细胞和整体水平上阐明OPN诱导VSMC迁移的分子机制和信号途径，揭示整合素β3-FAK活化在血管病变中的意义，为寻找可防止血管内膜增生的新靶标提供理论依据。一、细胞外基质诱导血管平滑肌细胞迁移及其与整合素β3和粘着斑激酶表达的关系细胞-ECM之间的相互作用是VSMC粘附、迁移、增殖和分化的分子基础，这种相互作用是由ECM蛋白受体——整合素所介导的。因此，本研究从细胞-ECM相互作用的角度，探讨ECM蛋白（包括OPN、纤粘蛋白 fibronectin, FN）对VSMC迁移的影响及其与整合素β3和FAK表达之间的关系。实验结果如下:1. 用生长至完全汇合的VSMC进行平面迁移分析，结果显示，用5、10、20μg/ml OPN处理24h的VSMC, 其平面迁移活性分别为对照组（1.5% NCS）的1.04、1.36和1.57倍。FN在低浓度时（5、10μg/ml）不能诱导VSMC的迁移。但在高浓度（20μg/ml）时显示出一定的促迁移效应。随着OPN、FN作用时间的延长，VSMC的迁移距离也相应增大。提示，OPN和FN均具有促VSMC迁移的活性，其中以OPN的作用更为明显，并且在一定范围内具有量-效和时-效关系。2. Western印迹分析显示，从未经处理的VSMC中即可检测到低水平的整合素?3的存在。在被OPN、FN处理24h的VSMC中，整合素?3含量明显升高。两种ECM蛋白相比，OPN对整合素?3表达的上调作用明显大于FN，并且与OPN促细胞迁移的活性相一致。提示整合素?3可能参与了OPN、FN诱导的VSMC迁移过程。3. 提取不同实验组VSMC的总RNA，进行实时定量RT-PCR分析，检测OPN、FN对迁移相关分子FAK、Gax基因表达的影响。结果显示，未经OPN、FN处理的VSMC，其FAK基因转录活性较低，难于测及；而在用OPN、FN（20μg/ml）刺激3、6、12、24h的VSMC中，均可检测到较高水平的FAK mRNA，但未观察到明显的时间效应。此外，OPN和FN还可不同程度地抑制Gax基因表达，以刺激细胞12h时，Gax mRNA下降最为明显。其中OPN下调Gax表达的作用大于FN；24h<WP=6>后又恢复接近至正常水平。可见，两种ECM蛋白引起的Gax表达下调具有一过性的特征。由于Gax是细胞迁移的负调控因子，该基因的表达下调可能是OPN诱导VSMC迁移的另一分子机制。上述结果提示，OPN、FN通过诱导整合素?3的表达，上调FAK基因转录活性，抑制迁移负调控因子Gax的表达, 进而刺激VSMC的迁移。本实验证实，整合素β3-FAK是介导OPN和FN与VSMC相互作用并引发细胞迁移的信号转导途径之一。二、FRNK对OPN诱导血管平滑肌细胞迁移的抑制作用及其分子机制为进一步阐明整合素β3-FAK信号途径在骨桥蛋白（OPN）诱导血管平滑肌细胞（VSMC）迁移中的作用和意义，本研究通过转染FAK磷酸化激活的特异性抑制剂——粘着斑相关非激酶（FRNK）的表达质粒pGZ21δxZ-FRNK，选择性阻断FAK磷酸化，观察其对OPN-整合素β3相互作用所激活的信号通路的影响以及与OPN诱导VSMC迁移之间的关系。结果发现：1. 用Western blot分析检测外源性FRNK在VSMC中的表达情况。结果表明，细胞在转染pGZ21δxZ-FRNK重组子24h后，VSMC中的FRNK含量明显增加，约是未转染组的2.5倍；转染后36h，FRNK水?更多还原

【Abstract】 Migration of vascular smooth muscle cell (VSMC) from media to intima is an earlier event in response to vascular injury, such as atherosclerosis and restenosis after percutaneous transluminal coronary angioplasty (PTCA). VSMC migration is the biological basis of neointima formation. Many of growth factors, cytokines and extacellular matrix (ECM) are involved in the migration of VSMC toward intima. However, the signaling pathways that regulate VSMC migration are still obscure.Recent findings have shown that integrins, as ECM receptors, not only mediate the interaction of ECM-cell and cell-cell, but also integrate extracellular stimuli, then transmitte the signal into cell. Focal adhesion kinase (FAK) is the intermediator linking integrins to downstream signal proteins, and plays a key role in multiple signal pathways. FAK recruits signal proteins such as PI-3K by means of its tyrosine sites, and then results in cascade phosphylation, thereby allowing signals to transfer further into cell. The signaling pathway is required for regulating cell contraction and movement.Our previous studies have demonstrated that VSMC in vitro cultured and in arterial neointima can secrete a substantial ECM, including osteopontin (OPN) and fibronectin (FN). As the marker of synthetic VSMC, OPN promotes adhesion of VSMC, through interaction with integrinβ3, by which cell is anchored in ECM. However the signal pathway triggered by OPN in VSMC migration isn’t well understood. Although many studies focus on the roles of integrin signal pathway in regulation cell migration, it cann’t still determine whether integrinβ3-FAK is implicated in OPN-induced migration of VSMC. In present study, we characterized the effect of OPN on VSMC migration and investigated the relationship<WP=12>between OPN-induced VSMC migration and the activation of integrinβ3-FAK signal pathway. By transfecting the recombinants of FAK related non-kinase (FRNK) into VSMC to inhibit FAK activation, we try to confirm whether integrinβ3-FAK signal pathway plays important roles in OPN-induced VSMC migration. 1. Relationship between expression of integrinβ3, FAK and migration of VSMC stimulated by ECMThe interaction of cell-ECM is the molecular basis of VSMC adhesion, migration, proliferation and differentiation, this interaction is mediated by ECM receptor integrins. In the study, we investigated the effect of ECM proteins (including OPN and FN) on VSMC migration and the relationship between expression of integrinβ3, FAK and migration of VSMC stimulated by ECM. The results as following:(1) VSMC migration was examined using a model of wounding injury of confluent cells. The migration activity of VSMC stimulated by OPN (5, 10, 20μg/ml) was increased by 1.04, 1.36 and 1.57 folds, compared with control VSMC, respectively. FN promoted migration of VSMC only in high dose (20μg/ml), but not in low dose. The data indicated that both of OPN and FN induced VSMC migration in dose-dependent manner, OPN was more effective than FN.(2) The results of Western blotting suggested that there was low expression level of integrinβ3 in untreated VSMC, but expression of integrinβ3 was greatly upregulated after treated by OPN and FN for 24h. The upregulation of integrinβ3 induced by OPN was more effective and was paralleled to migration induced by OPN. We speculated that integrinβ3 might be involved in VSMC migration induced by OPN and FN.(3) To test the effect of OPN and FN on the expression of migration related molecules FAK and Gax, the two genes were detected by real time RT-PCR. The mRNA level of FAK could not be detected in untreated VSMC. After stimulated by OPN and FN (20μg/ml) for 3, 6, 12 and 24h, the mRNA level of FAK was greatly increased, but not in time-dependent<WP=13>manner. In addition, OPN and FN could also inhibit expression of transcription factor Gax, respectively. OPN was more effective than FN. Because Gax is capable of inhibiting cell migration, downregulation of Gax induced by OPN might be one of molecular mechanisms of OPN-induced VS更多还原