DNA双链断裂对颈动脉再狭窄过程中平滑肌细胞增殖与迁移的作用

项目名称： DNA双链断裂对颈动脉再狭窄过程中平滑肌细胞增殖与迁移的作用

项目编号： No.81470587

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 医药、卫生

项目作者： 罗涛

作者单位： 首都医科大学

项目金额： 73万元

中文摘要： 颈动脉狭窄是缺血性脑血管病的重要病因，颈动脉支架成形后再狭窄问题仍是亟待解决的远期并发症，血管平滑肌细胞(VSMC)增殖和迁移对再狭窄过程起关键作用，但其分子机制尚未研究清楚。目前研究发现机体存在识别修复DNA损伤的能力，但发生严重的DNA双链断裂(DSBs)产生的生物学毒性可导致其异常复制和细胞增殖，我们在前期工作中发现人颈动脉再狭窄病变中存在DSBs应答与修复,Uhrf2基因参与该应答过程。据此提出DSBs应答可能参与颈动脉再狭窄中VSMC单克隆增殖与迁移的过程假设。我们将采用大鼠颈动脉球囊损伤模型，检测DNA损伤应答验证检查点失活和DNA修复途径，通过激光照射VSMC诱导DSBs方法，结合逆转录病毒载体、shRNA基因敲除和GFP示踪技术，探讨DSBs应答对VSMC增殖通路的影响和microRNA的介导作用，揭示颈动脉再狭窄和DNA损伤的潜在关系和规律，为动脉治疗新靶点奠定基础。

中文关键词： 再狭窄；血管平滑肌细胞；DNA双链断裂；细胞周期检查点；微小RNA

英文摘要： Carotid stenosis is a narrowing or constriction of the inner surface of the carotid artery, and a major risk factor for stroke that leads to brain damage. In the past decades, carotid angioplasty and stenting (CAS) has been developed into a credible option for the patients with carotid stenosis. However, restenosis remains a severe and unsolved issue after CAS treatment. Restenosis is the arterial wall's healing response to mechanical injury, and characterized by neointimal hyperplasia, which is partially caused by vascular smooth muscle cells (VSMC) proliferation/migration. However, the molecular mechanisms involved in the restenosis are still largely unclear. Emerging evidences have supported a crucial role of DNA damage response in the development and progression of atherosclerosis, which is caused by the the accumulation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). Interestingly, increased oxidative stress is also considered as a major characteristic of restenosis following CAS treatment, which plays an important role in neointima formation. It has been demonstrated that oxidative DNA adduct, 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG), and DNA repair enzyme, PARP1, are quickly increased in carotid arteries following carotid angioplasty. Among the mechanisms that cells have developed to cope with the constant DNA lesions are elegant but not perfect DNA-repair processes. As the most serious one, DNA double-strand breaks (DSBs) do not occur frequently, but they are extremely toxic and difficult to be repaired, which also interfere with cell cycle checkpoint activation. Our preliminary study suggests that DSBs and DNA repair occurred in the carotid artery with restenosis, especially in the thickened neointima area. Using laser micro-irradiation, we found that Uhrf2 was recruited to the sites of DNA damage, so it is likely to act as a dominant role in mouse VSMC to maintain genomic stability. Above all, we hypothesized that DSBs response and repair participate into the process of carotid restenosis by activation of VSMC monoclonal proliferation/migration. We will use carotid artery balloon injury rat model to analyze the activation of DNA damage repair pathways and cell cycle checkpoint in the carotid restenosis at various levels (animal vivo and vitro, molecule, gene). And laser micro-irradation induced DSBs of VSMC, combined with knockout gene by shRNA to explore impact of VSMC proliferation/migration of DSBs damage and response, and to uncover molecular pathways of VSMC proliferation/migration related DNA response and repair with microRNA-mediated effects. This study may provide clues for developing novel therapeutic strategies for carotid artery restenosis following CAS treatment.

英文关键词： Restenosis;Vascular smooth muscle cell;DNA double strand break;Cell cycle checkpoint;MicroRNA