应用iPS细胞研究马凡综合征主动脉平滑肌细胞凋亡的发生机制及相关药物筛选

Aortic dissection and aneurysm is the most fatal complication clinically in patients with Marfan syndrome. However, the mechanism of Marfan syndrome and related prophylactic treatment remains unclear. Previous studies have shown that the cystic degeneration of the aortic smooth muscle cells in Marfan syndrome patients is mainly due to apoptosis of vascular smooth muscle cells (VSMC), dissociation of elastic tissue and accumulation of mucin and fibrin. Our previous work also showed that in mouse models of Marfan syndrome, middle aortic elastic tissue was found fractured, the apoptosis of VSMC increased and MMP-2, AngII, TGF-β and other factors upregulated in VSMC. In order to define the effect of drug therapy on the apoptosis of VSMC, we will establish the models of Marfan syndrome using disease-specific human induced pluripotent stem cells (hiPSC) at the cellular level for observing the apoptosis of the middle smooth muscle and the expression of associated signaling pathways. For the purpose of evaluating the improvement on the apoptosis of smooth muscle cells of different drugs, the differentiated cells from the use of ACEIs, ARBs and doxycycline drug treatment, through ATII, TGF-β, MMP2 signaling pathway regulating explore appropriate muscle apoptosis improve the situation, with a view to improving the evaluation of different drugs on patients with Marfan smooth muscle cell apoptosis in the case. The results of this study will not only help reveal the effect of prophylactic therapy on the improvement of vascular smooth muscle apoptosis in Marfan syndrome, but also provide a brand new platform for the disease model and the study of molecular mechanisms of Marfan syndrome.

主动脉夹层和动脉瘤是马凡综合征患者临床上最为致命的病变，但对于该病变的发生机制以及相关的药物预防性治疗，目前还尚不明确。我们之前的研究工作显示，在小鼠的马凡疾病模型上发现主动脉中层弹性组织断裂，中层组织平滑肌细胞凋亡增加，平滑肌细胞中MMP-2，AngII，TGF-β等因子表达上调。为明确药物对于平滑肌细胞凋亡的作用，本研究拟利用疾病特异性的人类诱导多能干细胞，建立马凡综合征血管病变在细胞水平上的疾病模型，以观察马凡患者平滑肌细胞中相应通路的表达和细胞凋亡情况；其后对分化而来的细胞分别运用ACEIs、ARBs以及强力霉素等药物处理，通过对ATII、TGF-β、MMP2信号通路的调节，探索相应的平滑肌细胞凋亡改善情况，以期能够评价不同药物对马凡患者平滑肌细胞凋亡的改善情况。本研究的结果不仅有助于揭示药物预防性治疗对于马凡综合征血管平滑肌凋亡的改善作用，还能提供iPS这一新的研究平台。

马凡综合征多表现为主动脉根部以及升主动脉近端的瘤样扩张，而较少累及远端。这可能与血管中层不同胚层来源平滑肌受致病基因影响程度不同有关。诱导多能干细胞可以保留患者的突变的基因信息且可以控制分化条件定向分化为不同胚层来源的血管平滑肌细胞。我们获取患者和健康人的体细胞，并诱导重编程为iPSC，建立马凡综合征疾病和健康对照组iPSC细胞系，诱导 iPSC 分化为血管平滑肌细胞，并验证其胚层来源特异性和表型的差异，证明诱导分化的平滑肌细胞具有马凡综合征的表型特点。用所建立的马凡综合征疾病模型研究动物模型中取得成效的药物对平滑肌细胞凋亡改善情况，并探索用疾病模型进行新药研发或药物筛选方面应用前景。现已建立6例临床诊断马凡综合征患者iPSC，其中4例进行全外显子测序，3例FBN1突变，1例FBN2突变。并在分化所得血管平滑肌模型中发现，主动脉根部平滑肌细胞更肥大，且肌纤维更加粗大，肌纤维排列的更加杂乱无序，凋亡水平较正常组高。基质金属蛋白酶抑制剂强力霉素可以有效的降低马凡综合征患者血管平滑肌的凋亡水平，降低细胞外基质的降解。此项目提供了马凡综合征等遗传性心血管疾病的iPSC研究技术平台，并筛选了强力霉素这一将来可能运用于临床以减缓马凡综合征患者主动脉相关并发症发展的药物。