ATM通路介导的DNA损伤应答在肺血管重构中的作用与机制研究

Pulmonary vascular remodeling is one of the major pathological characteristics of pulmonary arterial hypertension (PAH), of which the vascular smooth muscle cells (VSMCs) proliferation and migration are generally accepted as the key points. Consequently, understanding the mechanisms of VSMCs proliferation is the key to inhibit and reverse pulmonary vascular remodeling. Recent foreign studies suggested that 1) VSMCs in PAH exhibited cancer-like characteristics, including self-sufficiency in growth signals, insensitivity to anti-growth signals, evading apoptosis, and cellular metabolic disorder, which gave rise to the VSMCs abnormal proliferation property; and 2) somatic chromosome abnormality, a kind of genomic instability, existed in patients with PAH, suggesting a correlation between PAH development and genomic alteration. Hence it follows that the abnormality of maintaining stability of the genome and correct transmission of the genetic information, a popular topic in cancer study, could be involved in the aetiology of vascular remodeling in PAH. In other words, the mechanism that mediates VSMCs abnormal proliferation is likely to be extremely similar to that of cancer. To make it more clear, we focus on the DNA damage response (DDR), a regulation mechanism which is of crucial importance to maintain normal physiological function and stable genetic characters in cells. In addition, abnormality of this essential regulatory system may be closely related to carcinogenesis and genomic instability. To our knowledge, there is no report on the role of DDR mechanism in pulmonary vascular remodeling by now. We propose that impairment of the DDR system which is led by ATM signal pathway induced the VSMCs abnormal proliferation, while normal ATM can make cell cycle arrest through ATM-Chk2-p53 pathway and may participate into DNA damage repair, exerting temporal and spatial control of repairing DNA damage respectively. In the present project, we plan to 1) set up the rat model of PAH by monocrotaline injection after unilateral pneumonectomy,; and 2) preliminarily elucidate the roles of ATM-Chk2-P53 cell cycle arrest pathway in the abnormal proliferation of VSMCs by questing the temporal expression of the key proteins in the signal pathway. Through these studies, we hope to establish the theory base of PAH treatment and improving patients' life quality, then to find new targets for preventing and reversing PAH.

对血管平滑肌细胞（VSMCs）增殖机制研究是防治和逆转肺血管重构的关键。研究表明，VSMCs的增殖过程存在和肿瘤细胞相似的特性，因此，我们重点关注DNA损伤应答（DDR）这一与肿瘤细胞增殖和基因组不稳定性密切相关的调控网络，目前DDR在肺血管重构中的研究尚属空白。我们前期发现在肺血管重构过程中ATM蛋白激酶介导的DDR表达存在"拐点"现象，因此推测ATM通路介导的DDR的受损是推动VSMCs过度增殖的重要因素。本课题研究拟观察不同时期肺动脉高压大鼠模型中DDR中的关键通路ATM-Chk2-P53在VSMCs增殖过程中的时空表达，为防止和逆转肺动脉高压血管重构提供新靶点。

本项目的研究目标为1) 回答DNA 损伤应答机制在肺动脉高压发病过程中的作用；2) 探讨ATM 信号通路在以VSMCs 增殖为代表的血管重构中的时间轴表达。按照项目计划书的要求，我们建立了肺动脉高压大鼠模型并对DNA损伤应答机制中的ATM信号通路进行了研究。具体研究结果如下：.1. 成功建立了肺动脉高压大鼠模型。利用单侧肺切除手术加野百合碱的方法，建立大鼠肺高压模型，术后35天组的大鼠，其肺动脉压力显著增高，HE染色显示肺部血管出现平滑肌增厚、小血管肌化等变化，证明模型建立成功。.2. ATM信号通路在血管重构中的作用。我们选取术后21天、35天和49天组大鼠，分别观察ATM、磷酸化ATM、Chk2、P53、磷酸化P53以及P21等蛋白的变化。免疫组化的结果显示，与对照组相比，ATM、磷酸化ATM、Chk2以及P21在21天和35天组增加而在49天组较前两组下降。P53和磷酸化P53则在21天组有明显增加后而在35天及49天组下降。荧光定量PCR的结果同样显示，ATM, Chk2, P53在21天和35天组中的表达相比对照组显著增加(P<0.05)，在49天组中的表达相比前两组显著降低(P<0.05)。证实了我们提出的“ATM表达折点假说”，发现单侧肺切除手术加野百合碱制作的肺动脉高压大鼠模型中ATM表达折点在28-35天。在此之前，ATM信号通路呈高表达，在此之后，表达明显受抑制，影响DNA损伤应答的修复过程。这部分结果已完成英文文章撰写，正在投稿。.3. 利用转录组测序的方法，我们对建模后14天、28天和49天组的模型大鼠与对照组间差异表达的基因进行了分析。结果显示，模型组与对照组共有23个共有的差异表达基因，包括ATM信号通路下游的基因 TCTP、硬脂酰辅酶A脱氢酶SCD-1、BPIFB1等均有一致性的折点样表达，再次证实ATM信号通路的表达呈折点样改变。结果与本项目的研究预期相符，对于印证这些基因在肺动脉高压中的作用及机制研究将成为后续研究的重点。.综上，我们如期完成了项目计划书上的项目，研究结果与我们的设想一致，充分证实了我们提出的ATM-Chk2-p53-p21途径从量到质的转化，是引起细胞周期监测点功能及激活DNA修复复合体信号通路失活，导致了重度肺动脉高压时VSMCs过度增殖的重要原因这一折点假说。我们将在后续工作中对其调控机制作深入研究。