Rac1的新亚型Rac1b在体内和体外血管平滑肌细胞增生及迁移中的作用

We first discovered and cloned Rac1b, a new subtype of Rac1, in human aortic smooth muscle cells (VSMC) cDNA library. Rac1b is a splicing varient of Rac1 with an extra exon 3b and plays an important role in the pathogenesis, invasion, and metastases of the breast, colon and lung cancer. In our pilot studies, we expressed and purified recombinant Rac1b from Ecoli and successfully made its specific antibodies. As shown by Western blotting, Rac1b is expressed in cultured human aortic (Ao184) and human coronary artery (Co396) VSMC. Surprisingly, it is highly expressed in rat left carotid artery at 4 and 12 weeks after balloon injury, but it is not detectable in the right carotid artery and in the aorta. Immunohistochemistry shows the expressed Rac1b is limited to the balloon injury-induced neointima. In protein pull-down experiment, not like Rac1, Rac1b from the rat neointima lysate binds to its downstream PAK-1 (protein Cdc42/Rac1-activated kinase 1) in a GTP-independent manner, indicating Rac1b is constitutively active in vivo. These results strongly indicate Rac1b's up-regulation is closely related to VSMC proliferation and/or migration. Next, we plan to: 1) establish a 1-16 week full time course of Rac1b's up-regulation in rat neointima; 2) test whether blocking Rac1b's expression prior to or at the peak of the up-regulation could retard the neointima formation in vivo; 3) study whether Rac1b's mainly regulate VSMC's proliferation and/or migration; 4) search for the pathological stimulants for Rac1b's up-regulation in cultured VSMC; and 5) determine the signal transduction pathway of Rac1b in VSMC. Our study aims to investigate whether Rac1b is a new drug-discovery target for effectively blocking neointima formation and restenosis after coronary artery intervention injury.

申请人在人血管平滑肌细胞（VSMC）中偶然新发现并克隆了小GTP结合蛋白Rac1的新亚型Rac1b。Rac1b是Rac1的多了一个外显子3B的剪接变异体，与乳腺结肠及肺肿瘤的发生与转移关系密切。我们已成功自制了优质特异抗体。预实验发现Rac1b在在体大鼠主动脉中无表达、在培养的人主动脉及冠状动脉VSMC中有表达、在损伤后第4周及第12周的大鼠颈动脉中高表达、免疫组化显示高表达的Rac1b局限在新生内膜中、蛋白质下拉实验显示新生内膜中的Rac1b具有GTP非依赖性的永久活性，提示其表达与VSMC增生和/或迁移密切相关。下一步，本课题要完成其在新生内膜中表达增高的1-16周时间曲线；然后在最佳时间点抑制其表达后观察新生内膜的变化；解明其是调节VSMC增生还是迁移；寻找是什么生长/细胞因子使其表达上调；并探索其促进VSMC增生和/或迁移的信号通路。从而试图找出能阻止血管损伤后管腔再狭窄的新药靶。

近年我国心脑血管疾病的发病率和死亡率呈迅速上升趋势，每年造成沉重的经济损失和社会负担。血管平滑肌细胞（VSMC）异常增生贯穿动脉粥样硬化整个发生发展过程，尤其在冠脉支架安置术后新生内膜的形成而造成再狭窄中扮演着关键角色。因此，急需解明血管损伤后VSMC异常增生的分子机理。申请人在VSMC中偶然新发现并克隆了Rac1的新亚型Rac1b。Rac1b是Rac1的多了一个外显子3B的剪接变异体，与乳腺癌、结肠癌及肺癌的发生、转移及预后关系密切。.我们取得的主要结果有：.1）.Rac1b在在体WT型大鼠主动脉中无表达、在培养的人VSMC中有表达。在大鼠颈动脉损伤后第1周就开始上调，维持到16周。.2）.与WT大鼠相比，Rac1b-KO大鼠的颈动脉损伤后的新生内膜厚度有显著性降低。.3）.用慢病毒感染HEK293T细胞后观察到Rac1b有显著促进细胞迁移及增生，抑制细胞凋亡的双重作用。而对于VSMC的研究，实验进行得不是很顺利，可能与没病毒导致的Rac1b超表达浓度过高，细胞不能耐受，生长因此受到影响。为了克服这一困难，我们又制作了Tagln启动子驱动的VSMC特异性超表达Rac1b的大鼠。目前正在分离这些大鼠的VSMC进行原代培养，然后比较功能的差异。.4）.Rac1b促进增生是通过上调Cyclin-D1来实现的，而其抗凋亡作用是通过抑制Caspase-3来完成的。 为了进一步研究超表达Rac1b对基因表达的影响及寻找新的信号通路。我们在HEK293细胞上分别转染LV-Ctl、LV-Rac1及LV-Rac1b三种慢病毒后，进行表达谱芯片分析，发现了482~505个差异表达基因。确定了其通过JNK2/c-JUN/Cyclin-D1通路促进增生，通过AKT2/MCL1 通路抑制凋亡。.5）.为了在VSMC细胞中寻找新的作用通路，我们也进行了同样的病毒感染及芯片分析，发现约124个差异表达基因，结果正在分析验证中。.6）.我们将上述二种细胞的表达谱芯片进行综合分析，筛选出124个重点基因。在从WT，Rac1b-KO，Rac1b-TG三种大鼠中分离出的主动脉平滑肌细胞中进一步验证。预计可以找出Rac1b在VSMC中的作用通路。.总之，本课题结果提示Rac1b可能是阻止血管损伤后由于VSMC细胞异常增生导致管腔再狭窄的新药靶。