14-3-3γ-PCNA交互作用在VSMC增殖与凋亡中的功能与机制研究

Coronary artery bypass grafting is the gold standard for the treatment of coronary artery disease and ischemic cardiomyopathy, but vein graft often occurs restenosis after transplantation, which leads to graft failure. Therefore, the prevention of graft restenosis is of great significance to clinical trials. Abnormal proliferation and intimal hyperplasia of VSMC are the key factors leading to restenosis. PCNA is the most relevant protein for VSMC proliferation. In the early stage, we drew the protein spectrum of PCNA binding ligand by proteomic method. It was found that 14-3-3γ protein, which plays an important role in blood vessels, is one of PCNA binding ligand proteins, and they all works through post-translational modification. Therefore, we hypothesized that 14-3-3γ and PCNA were phosphorylated to form complex and regulate VSMC proliferation and apoptosis. We intend to study the mechanism of 14-3-3γ-PCNA interaction regulating VSMC in vein graft restenosis, construct 14-3-3γ, PCNA overexpression/interference lentiviral vectors, and conduct a series of studies in human aortic smooth muscle cells to observe the biological effect and regulation mechanism of 14-3-3γ and PCNA interaction in smooth muscle cell proliferation and apoptosis. At the same time, it will be further researched in the whole animal model.

冠脉旁路移植术是治疗冠脉血管疾病、缺血性心肌病的金标准，而静脉移植术后常常发生再狭窄导致移植失败，因此，防治移植再狭窄具有重要的临床意义。VSMC异常增殖和内膜增生是导致再狭窄的关键环节，PCNA是VSMC增殖最为相关的蛋白。前期我们通过蛋白组学方法绘制PCNA结合的配体蛋白谱，发现在血管中具有重要作用的14-3-3γ蛋白是PCNA的配体蛋白之一，且它们均通过翻译后修饰发挥功能。因此我们提出假设14-3-3γ与PCNA发生磷酸化修饰，形成复合体并共同调控VSMC的增殖与凋亡。我们拟开展14-3-3γ-PCNA交互作用调控VSMC在移植静脉再狭窄中的机制研究，构建14-3-3γ、PCNA过表达/低表达慢病毒载体，在人主动脉平滑肌细胞中开展一系列研究，观察14-3-3γ与PCNA交互作用在平滑肌细胞增殖与凋亡中的生物学效应及其调控机制；同时在整体动物模型中进一步研究论证。

冠脉旁路移植术是治疗冠脉血管疾病、缺血性心肌病的金标准，而静脉移植术后常常发生再狭窄导致移植失败，因此，防治移植再狭窄具有重要的临床意义。我们前期研究发现14-3-3γ蛋白是移植静脉重塑中PCNA重要的配体蛋白，是一个全新的发现，据此开展了14-3-3γ-PCNA交互作用调控VSMC在移植静脉再狭窄中的机制研究，发现：①14-3-3γ/PCNA可抑制VSMC细胞凋亡，提高PCNA磷酸化水平，促进细胞增殖；②PCNA可通过Bax/Bcl-2通路影响VSMC细胞增殖和凋亡；③PDGF-BB诱导的VSMC中miR-200a-3p表达量明显下降，miR-200a-3p靶向负调控14-3-3γ信号，过表达miR-200a-3p可抑制PDGF-BB诱导的VSMCs细胞增殖及表型转换。我们的结果表明miR-200a-3p/14-3-3γ/PCNA信号可能是移植静脉再狭窄的关键信号，为静脉移植失败提供了新的药物治疗靶点。