GTF2H4在心脏微血管内皮细胞缺血再灌注损伤中的关键作用及分子机制研究

Ischemia-reperfusion injury is a common cardiovascular disease with a high mortality rate. At present, it is mainly treated according to patients’ clinic symptoms. In recent years, it has been found that microcirculation injury could be obviously improved through protecting microvascular endothelial cells, but the mechanism is still unclear. Previous study indicated that GTF2H4 had the role of nucleotide excision repair, RNA transcription and regulation of cell cycle. Therefore, GTF2H4 is presumed to participate in repair the injury of microvascular endothelial cells. DNA nanotechnology technology has the advantages of intelligent, target and no side effects to regulate the transcription of GTF2H4. The study is designed to study GTF2H4 and its mechanism on the repair of ischemia reperfusion injury in cardiac microvascular endothelial cells, via detecting characteristics and analyzing the whole transcriptome of cells in various states. Then the DNA nanostructure-based delivery systems were applied to deliver microRNA and siRNAs of GTF2H4, so as to clarify the regulation of the ischemic reperfusion injury in cardiac microvascular endothelial cells and reveal the molecular network. Finally, the data of the study could enrich and deepen the understanding of the biological function of GTF2H4 and establish the experimental and theoretical evidence of GTF2H4-based treatment on cardiac injury.

缺血再灌注损伤是常见心血管疾病，死亡率较高，目前仍以对症治疗为主。近几年研究发现修复损伤的微血管内皮细胞可以明显改善微循环，但其机制欠明。研究发现，GTF2H4 的作用是核苷酸切除修复，RNA转录和调控细胞周期，推测其可能参与微血管内皮细胞的损伤修复。借助DNA nanotechnology技术来调控GTF2H4的转录具备智能、靶向、无副作用的优势。本课题拟通过检测细胞的各种特性、分析不同状态的全转录组等来研究GTF2H4在心脏微血管内皮细胞缺血再灌注损伤修复中的作用与机制，再构建DNA纳米自组装体靶向递送GTF2H4的microRNA和siRNA，阐述其在心脏微血管内皮细胞缺血再灌注损伤修复中的调控作用，揭示参与其中的分子网络，从而丰富和深化对GTF2H4生物学功能的研究和认识，为深入开展以GTF2H4为基础治疗心脏损伤奠定实验和理论基础。

TFIIH亚基4在细胞生长、发育、代谢等方面发挥重要的调控作用。然而，GTF2H4参与缺血性疾病以及缺血再灌注损伤病理过程的具体生物学机制还不明晰。实验设计对C57BL/6J雄性小鼠行冠状动脉左前降支结扎手术以实现心肌梗死造模，在梗死后第3、7天通过共聚焦显微镜观察到CD31、α-SMA双阳内皮细胞所占的比例明显增加，提示心肌梗死后内皮细胞发生间质转分化；对微血管内皮细胞HMEC-1行缺氧和血清剥夺处理1-3天，发现缺氧情况下微血管内皮细胞逐渐丢失VE-Cadherin、CD31等内皮细胞标志物，获得α-SMA、Fibronectin、FSP-1等间质细胞标志物，同时，检测到微血管内皮细胞中GTF2H4蛋白质水平随缺氧而逐渐下降，提示GTF2H4可能参与了缺氧调控的部分内皮间质化； GTF2H4过表达改善了缺氧诱导的细胞活力损伤，抑制凋亡，促进迁移，抑制小管形成，促进缺氧诱导的部分内皮间质化，而GTF2H4敲低在微血管内皮细胞上呈现出相反的效应；通过4D label free蛋白质组学分析和Western Blot及RT-qPCR验证得到GTF2H4通过调控自噬介导的蛋白质降解路径正向调控其毗邻分子ERCC3。通过4D- abel free磷酸化蛋白质组学富集分析得到NF-κB可能参与了GTF2H4调控的部分内皮间质化，并利用NF-κB磷酸化抗体芯片、双荧光素酶试验和凝胶迁移实验进行了验证，得到GTF2H4促进NF-κB通路激活的结果。磷酸化蛋白质组学的数据发现NCOA3第1330位丝氨酸的磷酸化可能参与了GTF2H4调控的NFκB通路激活，通过点突变实验，验证了GTF2H4能够促进NCOA3 1330S的磷酸化，继而促进其与p65的结合和NF-κB转录激活；最后，观察下肢缺血术后血流再灌注情况，得到GTF2H4促进缺血后下肢血流恢复，伴随冰冻组织切片免疫荧光检测到CD31、α-SMA双阳毛细血管密度增加，而GTF2H4敲低呈现出相反的结果。最后利用DNA纳米子自组装体于在体层面动物模型上得到验证，纳米自组装体可精确靶向GTF2H4基因，干预GTF2H4转录，抑制部分内皮间质化，抑制组织缺血后的血管新生。以上揭示了GTF2H4参与调控缺血缺氧和再灌注损伤过程的潜在分子机制和信号网络，利用DNA纳米子自组装体实现GTF2H4 microRNA或siRNA靶向递送。