血小板来源的TET2对血管新内膜形成的作用及机制研究

Neointimal hyperplasia is an important factor in vascular restenosis leading to acute cardiovascular disease. However, most of the major studies focus on internal factors such as vascular smooth muscle cells (VSMC) itself, and we have little known about the effect of external factors such as platelets on VSMC. In the early stage, we have reported that activated platelets can enter VSMC, and transfer their miR-223 targeting PDGFR β to inhibit the proliferation of VSMC, suggesting that platelets play an important role in the regulation of vascular function homeostasis. In recent in vitro pre-experiments, we found that (1) activated platelets could promote the differentiation of VSMC, and the effect was more obvious than that of vesicles secreted by platelets; (2) TET2 expression in platelets was much higher than that in VSMC; (3) platelets-derived TET2 could be transferred to VSMC. Therefore, we suggest that activated platelets promote the differentiation of VSMC by transferring TET2 and play a protective role in neointimal hyperplasia. The aim of this study was to establish a model of co-culture of cells in vitro and the atherosclerotic femoral artery injury model of TET2 in platelet-specific knockout mice. Immunofluorescence, histopathology, molecular biology and other techniques were used to elucidate the protective effect of platelet transfer TET2 on neointimal hyperplasia and its mechanism, and to provide a new target for the accurate treatment of neointimal hyperplasia.

新生内膜形成是血管再狭窄而导致急性心血管疾病的重要因素。然而，大多数主要研究其内部因素即血管平滑肌细胞（VSMC）本身，外部因素如血小板对VSMC的作用研究较少。前期我们已发表活化血小板可进入VSMC，并转移其miR-223靶向PDGFRβ抑制VSMC增殖，提示血小板在血管功能稳态方面发挥重要调控作用。近期体外预实验我们发现①活化血小板可促进VSMC分化，且较血小板分泌的囊泡作用更明显；②血小板中含有TET2，且远高于VSMC；③血小板的TET2可转移至VSMC。因此，我们提出活化血小板通过转移TET2促进VSMC分化，对新生内膜形成发挥保护作用。本项目拟通过对体外细胞共培养及TET2在血小板特异性敲除小鼠的动脉粥样硬化股动脉损伤模型的建立，采用免疫荧光、组织病理学、分子生物学等技术手段，深入阐明血小板转移TET2对新生内膜形成的保护作用及其机制，为新生内膜形成的精准治疗提供新靶点。

根据我们的前期研究，在血管损伤的情况下，血小板对维持血管稳态具有非常重要的作用。然而，血小板对新生内膜形成的作用及其机制尚未完全明确。因此，深入了解有助于发现新的有效靶点，进而开发更有效地靶向治疗，具有重要的临床意义。该项目的主要研究内容：证明人的血小板TET2转移至人冠状动脉平滑肌细胞（HCASMC），并通过DNA去甲基化调控血管平滑肌细胞（VSMC）表型转换；利用TET2特异性敲除小鼠，进一步证明血小板TET2转移至VSMC的作用；在动物水平进一步确定血小板来源的TET2在血管损伤后新内膜形成的作用。研究结果表明，在没有细胞核的血小板中存在丰富的去甲基化转移酶TET2，并且其含量要比在VSMC中高很多。进一步研究发现，在小鼠血小板特异性敲除TET2后，血小板的功能基本不受影响，提示TET2在血小板的作用不在于调控血小板的重要功能。其次，将静止和激活的血小板分别与VSMC共培养，只有激活的血小板可以通过对关键分化基因去甲基化而促进VSMC分化。为了证明活化的血小板是通过转移TET2发挥促进VSMC分化的作用，申请人发现血小板特异性敲除TET2的血小板明显较正常血小板促进VSMC分化的能力减弱，而血小板特异性过表达TET2的血小板能增强促进VSMC分化。最后，在小鼠体内模型进一步验证在细胞水平的血小板来源的TET2对VSMC的作用。在小鼠股动脉损伤模型中，血小板特异性敲除TET2后股动脉的新内膜形成明显加重；然而，血小板特异性过表达TET2能显著抑制新内膜形成。该项目更进一步深入了解血小板对新生内膜形成的作用及其机制，为治疗动脉粥样硬化引起的新生内膜形成提供新的靶点。

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