【作者】 程云会；

【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， 2004， 博士

【摘要】 细胞骨架(cytoskeleton)是由多种结构蛋白和收缩蛋白按照特定的模式有机组装而形成的一种立体网格结构，是保持特定细胞形态和行使收缩功能的重要结构基础。细胞骨架包括微丝、微管和中间纤维三种形式，其中由肌动蛋白及其结合蛋白组成的微丝（丝状肌动蛋白，F-actin）是血管平滑肌细胞(vascular smooth muscle cells，VSMC)中细胞骨架的主要结构形式。由微丝聚集成束，再与肌球蛋白纤维（粗肌丝）结合共同组成细胞收缩装置——应力纤维。微丝本身是一种复杂的动态性网络结构，可随着细胞表型及功能的改变而发生可逆性聚合和解聚。许多微丝结合蛋白在该过程中起组织、调节作用。SM22α (smooth muscle 22 alpha, SM22α)是一种经典的VSMC标志蛋白，但迄今，对该蛋白的确切功能尚不清楚。一些体外实验证实，纯化的SM22α蛋白可与肌动蛋白单体以1:6的比例结合；而且发现在VSMC中，SM22α与平滑肌α-肌动蛋白（SM α-actin）共定位，故推测SM22α可能与VSMC微丝重构有关，但缺乏进一步的实验证据。为了证实SM22α在VSMC微丝重构、细胞收缩和运动中的作用，本研究利用血清饥饿诱导VSMC由合成型再分化为收缩型。首先观察VSMC表型逆转过程中，SM22α的表达变化规律及其与VSMC收缩和迁移等功能之间的关系。在此基础上，通过转染反义或正义SM22α表达载体，观察该基因表达变化对VSMC中的微丝重构和以此为结构基础的收缩和迁移等功能的影响，试图从分子、细胞和整体水平上探讨VSMC中SM22α的生理学意义及其作用的分子机制。1 SM22α对血管平滑肌细胞骨架及收缩功能的影响SM22α是收缩型VSMC的标志蛋白，为了探讨该蛋白与VSMC表型和功能的关系，本部分利用血清饥饿法诱导VSMC由合成型向收缩型转变，用RT-PCR对不同表型VSMC的SM22α表达活性进行检测，并通过转染反义SM22α表达载体，观察SM22α表达对VSMC细胞骨架和收缩<WP=5>功能的影响。实验结果如下：1.1 血清饥饿对SM22α基因表达的影响RT-PCR结果显示，在血清刺激培养的VSMC中几乎检测不到SM22α mRNA；血清饥饿24 h后，SM22α mRNA水平迅速上升至高峰，并一直维持此水平不变；血清饥饿72 h后的细胞再恢复血清刺激时，SM22α表达活性又快速下调。1.2 血清饥饿对收缩蛋白SM α-actin表达的影响Western blot分析结果显示，体外常规培养的合成型VSMC仍能表达较高水平的SM α-actin；血清饥饿后，SM α-actin的含量仅升高约1.5倍；再恢复血清刺激培养时，该蛋白又下降至基础水平，其表达模式与SM22α类似。1.3 SM22α表达对细胞骨架的影响采用改良考马斯亮蓝染色法显示细胞骨架，镜下可见血清饥饿可诱导VSMC细胞骨架由稀疏的网格状重构为致密的束状。转染反义pcD2-SM22α的VSMC血清饥饿后，其细胞骨架重构受阻。1.4 SM22α表达与VSMC收缩之间的关系 用乙酰胆碱刺激合成型VSMC时并不出现收缩反应。血清饥饿后，随着SM22α及SM α-actin的表达增加，VSMC也获得收缩能力，标志着细胞已转变为收缩型。转染反义pcD2-SM22α的细胞用乙酰胆碱刺激后，不再出现明显的细胞缩短现象。上述结果提示，SM22α仅在收缩型VSMC中表达，和SM α-actin等其他VSMC标志基因相比，前者更具有表型特异性。SM22α可能通过参与VSMC表型转变过程中细胞骨架的重构而影响细胞收缩功能的调节。2 在微丝重构过程中SM22α与SM α-actin之间的定位关系如前所述，微丝是细胞骨架的一种主要结构形式。为了进一步证实在微丝重构过程中，SM22α表达对微丝重构的影响及其与SM α-actin之间的定位关系，本部分分别以佛波酯(TPA)、蛋白激酶C (PKC) 抑制剂(GF109203X)和细胞松弛素(CB)作为微丝重构诱导剂，探讨了SM22α和SM α-actin在微丝中的重新分布及相关关系。结果如下：2.1 pEGFP-SM22α质粒在VSMC中的表达和产物定位在荧光显微镜下可观察到，转染pEGFP-SM22α的VSMC中，所表达<WP=6>的绿色荧光产物主要定位于核周围的胞浆中。免疫共沉淀结果显示，在血清培养的VSMC中，仅少量SM22α与SM α-actin结合；血清饥饿48 h后，与SM α-actin缔合存在的SM22α明显增多。2.2 SM22α过表达不影响TPA和CB诱导的微丝重构间接免疫荧光显示，血清饥饿培养48 h的VSMC中含有大量平行束状分布的微丝（应力纤维），用CB处理6 h后可使VSMC中的应力纤维消失，微丝全部解聚，呈弥散状分布于胞浆中。用TPA处理VSMC时，细胞中的应力纤维先解聚，形成podosome小体，而后参与形成胞膜皱褶。在TPA处理的转染pEGFP-SM22α的细胞中，可显示伴随着微丝重构过程，胞浆中的SM22α与SM α-actin共定位；并且在新形成的皱褶中，SM α-actin的出现早于SM22α。2.3 TPA、CB诱导的微丝重构过程中，伴有SM α-actin的重新分布胞浆/骨架蛋白分步提取分析显示，在血清饥饿48 h的VSMC中，SM α-actin多分布于细胞骨架中，其微丝与胞浆的蛋白含量之比（pellet/supernatant，P/S）约为1.32。用CB处理VSMC以破坏细胞中的应力纤维，可引起SM α-actin由微丝转向胞浆的重新分布，P/S下降为0.49。TPA虽可诱导VSMC 中的微丝发生重构，但对SM α-actin的重新分布影响不大，多数SM α-a更多还原

【Abstract】 The cytoskeleton of eukaryotic cells is a network structure composed of many kinds of structural and contractile proteins and plays an important role in maintaining cell shape and contraction. There are three types that include microfilaments, microtubles and intermediate filaments. The microfilaments (filamentous actin, F-actin) are a major kind of cytoskeleton in vascular smooth muscle cells (VSMC), which is assembled by actin and its binding proteins. The stress fibers (SF) are the contractile apparatus of VSMC, which is composed of microfilament bundles and myosin filaments (thick myofilaments). The microfilaments are a complex, plastic, dynamic network structure, which is associated with the shape and function changes of VSMC. Many proteins binding to actin play a regulatory role in microfilament remodeling progress.Smooth muscle 22 alpha (SM22α) is a 22-kD protein of smooth muscle-specific expression. Purified SM22α protein binds directly to actin filaments in a ratio of 1:6 monomers in vitro. SM22α and SM α-actin colocalize to stress fibers in cultured VSMC. Whether SM22α is a functional actin-associated protein in VSMC is controversial. In order to determine the role of SM22α in cytoskeleton remodeling, contraction and migration of VSMC, the model of VSMC redifferentiation induced by serum starvation was used in present study. We observed the changes of SM22α expression and its relation with cell contraction and migration during phenotype reversion of VSMC. After VSMC was transfected by recombinant SM22α antisense or sense plasmids, changes of the cytoskeleton structure and the ability of contraction and migration of VSMC were observed. It was investigated whether the expression of SM22α in <WP=11>VSMC could affect the cytoskeleton remodeling.1 Effect of SM22α on VSMC cytoskeleton and contraction SM22α is a marker of contractile VSMC. Here we observed the effect of SM22α on VSMC phenotype and function. During reversion of synthetic VSMC to contractile phenotype induced by serum starvation, the expression of SM22α gene in different phenotypes was detected by RT-PCR. To investigate the relationship between SM22α expression and cytoskeleton remodeling, VSMC was transfected by antisense SM22α plasmids, and then contraction of VSMC was evaluated. The results were as follows:1.1 As analyzed by RT-PCR, the mRNA of SM22α was almost undetected in VSMC stimulated by serum. The expression of SM22α was quickly upregulated following serum starvation and maintained at higher levels. In serum-restimulated VSMC after serum starvation for 72 hours, the expression of SM22α was down-regulated quickly again.1.2 Western analysis showed that the expression change of SM α-actin was consistent with SM22α. The level of SM α-actin protein increased about one and a half of folds in serum-deprived VSMC, and declined to the base level following serum restimulated.1.3 The VSMC cytoskeleton was shown by modified coomassie brilliant blue stainning. The cytoskeletal structure of VSMC was remodulated from discrete network to well arranged and dense bundles after serum starvation. This processe was blocked by tranfection of antisense plasmids.1.4 Acetylcholine (ACh) didn’t induce contraction of synthetic VSMC. When the expressions of SM22α and SM α-actin were upregulated, contraction of VSMC induced by ACh was found, suggesting that VSMC is capable of reversion from synthetic to contractile phenotype. However, the contraction induced by ACh was sustained in VSMC transfected by antisense SM22α recombinant. These data suggested that SM22α is only expressed in contractile VSMC. Compared with other VSMC markers, the expression specificity of SM22α is higher, suggesting that SM22α may play a role in regulation of VSMC <WP=12>cytoskeleton remodeling and contraction.2 SM22α colocalization with SM α-actin during microfilament remodelingIn order to identify the localization association between SM22α and SM α-actin during microfilament remodeling, VSMC was treated with the inducers, inc更多还原