Tudor-SN蛋白调控血管平滑肌细胞表型转化的机制研究

Phenotypic switching of vascular smooth muscle cell (VSMC) is considered as a leading cause for hemadostenosis in the cardiovascular disease. Very recently, we found that the expression level of multifunctional Tudor-SN protein in VSMC was associated with the proliferation ability of VSMC, and Tudor-SN could promote cell cycle progress and enhance proliferation ability of VSMC. The present project was designed to investigate the regulation mechanism of Tudor-SN in the VSMC phenotype transformation in terms of animal, cellular and molecular levels. 1) The expression levels of Smad3 or Sp1 were interfered to analyze the molecular mechanism of unregulated Tudor-SN during VSMC phenotype transformation.2) Tudor-SN-KO and Tudor-SN-WT C57BL/6N mices were utilized to study whether Tudor-SN has any effects on VSMC phenotypic switching after carotid artery injury in vivo. 3) PDGF was also used to induce the proliferation of VSMC, in order to evaluate the effect of Tudor-SN on the retraction, migration ability of VSMC. 4) To confirm the Tudor-SN-associated signal transduction pathway in the VSMC phenotype transformation, the PCR and western blotting assays were performed to validate the targeting genes. 5) ChIP assay of histone and CoIP were also performed to study the regulation mechanism of Tudor-SN in gene transcription.

血管平滑肌细胞（VSMC）表型转化（收缩型变为增殖型）是导致心血管疾病中血管狭窄的主要原因。近期我们发现多功能Tudor-SN蛋白在VSMC中的表达与细胞增殖能力相关,并促进VSMC的周期进程及增殖。本课题拟从动物、细胞和分子水平探讨Tudor-SN调控VSMC表型转化的机制：1)通过干扰Smad3或Sp1的表达，同时给予PDGF刺激，分析VSMC表型转化时Tudor-SN表达上升的调控机制；2) 利用Tudor-SN基因敲除小鼠及野生型小鼠建立动脉损伤模型，探讨Tudor-SN对损伤下VSMC表型转化的影响；3)利用PDGF诱导VSMC增殖，研究Tudor-SN对VSMC收缩能力和迁移能力的影响；4) 通过对筛选的靶基因进行PCR和WB的验证，明确Tudor-SN蛋白发挥作用的信号转导通路；5)荧光素酶实验、组蛋白ChIP及CoIP实验将揭示Tudor-SN调控靶基因转录的方式。

血管平滑肌细胞（VSMC）表型转化（收缩型变为增殖型）是导致心血管疾病中血管狭窄的主要原因。深入研究VSMC 表型转化及异常增殖的分子机制，有助于我们认识血管狭窄的发生发展过程，并为预防血管狭窄提供思路。课题组前期工作发现多功能Tudor-SN蛋白在VSMC中的表达水平与其增殖能力相关,并促进VSMC的周期进程及增殖。本课题拟探讨Tudor-SN在VSMC表型转化中的作用及调控机制。课题组构建了大鼠颈动脉和小鼠股动脉损伤模型，发现血管损伤后新生内膜中的VSMC中Tudor-SN蛋白表达水平显著升高。同时通过组织离体培养及PDGF诱导细胞等实验，进一步证实了VSMC发生表型转化时Tudor-SN蛋白表达上调。Tudor-SN蛋白的表达上调为其参与VSMC表型转化奠定了基础。随后我们通过生物信息学预测及细胞系内验证，证实了转录因子Elk1在血管损伤后Tudor-SN蛋白表达上调中发挥重要的转录调控作用。为了研究Tudor-SN蛋白对损伤后VSMC表型转化影响，我们构建了VSMC条件性敲除Tudor-SN小鼠，并构建股动脉损伤模型。结果显示，与WT小鼠相比，条敲小鼠血管损伤后新生内膜明显减少，血管狭窄显著降低。随后利用原代细胞及血管平滑肌细胞系，我们证实了Tudor-SN蛋白促进VSMC的增殖及迁移。机制研究证实，Tudor-SN蛋白作为转录共激活因子通过调控增殖及迁移相关的一系列基因促进VSMC的增殖迁移，调控其表型转化。本课题揭示了血管损伤后VSMC表型转化过程中Tudor-SN表达上调的调控机制，并证实Tudor-SN蛋白通过调控增殖迁移相关基因参与VSMC表型转化，帮助我们进一步认识疾病或损伤导致血管狭窄的具体机制，并为指导和改善床治疗效果提供新思路和理论依据。