Oct4下调Spry2表达在胸主动脉夹层血管平滑肌细胞表型转化中的作用及机制研究

The phenotype transition of vascular smooth muscle cells (SMCs) is a critical pathological process involved in the progression of thoracic aortic dissection (TAD). We found in our previous study that the over-expression of Oct4 significantly inhibited the level of α-SMA in pericardial interstitial cells (PICs), and thus prevented the differentiation of PICs into myofibroblast (MF). However, whether Oct4 exhibits a somewhat similar regulatory mechanism for the phenotypic modulation of vascular SMCs remains unclear. Our preliminary results in this project indicated that the over-expression of Oct4 in vascular SMCs promoted this alternation of SMCs phenotype abovementioned. Taking together with our finding that the 5'-untranslated region of Spry2, who acts as the inhibitor of receptor tyrosine kinase signal pathway and can modulate the phenotype of SMCs to a "contractile one", contains Oct4 recognition sites. We Speculated that Oct4 may introduce the phenotype transition of vascular SMCs to a "Synthetic type" by down-regulation of Spry2 level. In this project, we propose to evaluate the role of Oct4/Spry2 in the phenotype transition of aortic SMCs observed in TAD by multiple approaches including bioinformatic analysis, luciferase reporter assay, ChIP, adenovirus- (on lentivirus-) mediated gene delivery, SD rat TAD animal models and clinical investigations. Conceivably, all of these are conducive for the elucidation of underlining molecular mechanisms involved in the phenotype transition of aortic SMC observed in TAD and the progression of TAD is positive.

血管平滑肌细胞(SMC)表型转化是参与胸主动脉夹层(TAD)发生的重要病理过程。我们之前发现过表达Oct4可抑制心包间质细胞表达α-SMA并阻止其分化为肌成纤维细胞，但其对血管SMC是否具有类似的表型调控作用仍不明确。本项目前期研究结果发现在血管SMC过表达Oct4可导致上述表型转化。联系之前发现Spry2这一可使SMC向"收缩型"转化的受体酪氨酸激酶通路抑制蛋白的5'-非翻译区包含Oct4识别位点，我们设想：Oct4可下调Spry2的表达使SMC向"分泌型"转化并导致TAD发生。本项目拟通过ChIP、荧光素酶报告基因实验明确Oct4对Spry2转录的抑制作用，经细胞学实验明确Oct4/Spry2介导血管SMC表型转化的机制，并通过动物模型及临床标本研究明确Oct4/Spry2表达量与SMC表型转化及TAD发病的关联，对进一步揭示主动脉SMC表型转化及TAD发病的分子机制具有积极推动作用。

急性胸主动脉夹层（TAD）是一种致死性主动脉疾病。既往研究报道人主动脉平滑肌细胞（SMCs）在特定条件下发生去分化性表型转化是TAD发病的中轴性细胞学基础。OCT4是POU同源域转录因子家族的一员，在细胞自我更新过程中发挥关键调控作用，其在TAD主动脉壁的表达情况，SMCs表型转化中的作用机制及其与TAD发生发展的相关性未见报道。.本研究中我们通过临床TAD患者及大鼠TAD模型的主动脉组织经qPCR、western blot和免疫组化检测了OCT4及其下游靶基因SPRY2和KLF5以及SMCs表型标记分子的表达情况。原代培养人主动脉SMCs，通过细胞功能实验、染色质免疫共沉淀（ChIP）、荧光素酶报告基因明确在SMCs中OCT4对SPRY2和KLF5的转录调控作用；通过相关细胞功能实验明确OCT4下调SPRY2表达及上调KLF5表达促进人主动脉SMCs由终末分化的“收缩型”向分化程度相对较低的“合成型”转化的作用；经相关细胞功能实验明确OCT4通过调控MAPK通路活化状态进而影响SMCs表型转化。.与正常主动脉相比，人TAD及大鼠TAD模型主动脉组织中OCT4、KLF5的表达均显著升高，而SPRY2的表达显著降低，表型标记分子表达降低。细胞学实验结果显示，在SMCs中过表达OCT4可显著促进KLF5的转录表达，同时显著抑制SPRY2的转录表达。ChIP结果显示，OCT4蛋白可识别并结合于KLF5转录起始点上游-930至-922的ATTTGCCT序列，SPRY2转录起始点上游-569至-561的AAATTAAA序列。荧光素酶活性报告实验结果显示OCT4能够显著激活含野生型KLF5启动子序列的荧光素酶报告基因的转录，抑制含野生型SPRY2启动子序列的荧光素酶报告基因的转录，而OCT4结合位点突变的质粒则转录活性无显著变化。细胞功能实验结果显示，OCT4过表达能够促进SMCs表型由“收缩型”向表型标记分子表达降低而迁移能力增高的“合成型”转化。OCT4促SMCs表型转化的作用可以被SPRY2过表达、KLF5表达抑制或抑制p38蛋白磷酸化所阻断。.我们的研究结果提示，OCT4在TAD患者以及TAD大鼠模型的主动脉组织中表达上调。OCT4可通过下调SPRY2转录表达以及上调KLF5转录表达进而诱导SMCs由“收缩型”向“合成型”转化，该调控过程可能涉及p38通路的磷酸化激活。