PKM2/HIF-1正反馈环调节瘢痕疙瘩成纤维细胞的研究

Molecular mechanism of keloid remains to be elucidated. Recent studies have shown that keloids produce ATP through aerobic glycolysis, which has been found critical for cancer cells proliferation. Some factors participating in the glycolysis are closely related to the prognosis of some cancers and are considered to be potential target for anticancer therapy. Pyruvate kinase M2 (PKM2), interacting with HIF-1 and forming a positive feedback loop, plays an important role in regulation of cancer cells growth. Our previous studies found remarkable up-regulation of both PKM2 and HIF-1 in keloid fibroblasts and activation of PKM2, migrating from cytoplasm to nucleus. Therefore, we propose a hypothesis that the PKM2/HIF-1 positive feedback loop plays a key role in the regulation of keloids glycolysis and its pathologic proliferation. We would verify the hypothesis by regulating PKM2 expression and activity in keloid fibroblasts by gene engineering. Using ChIP-assay and Luciferase reporter gene assay technologies, this program may provide new insights into the interaction between PKM2 and HIF-1. Furthermore, with omics and gene rescue technologies, the regulation and molecular mechanism of PKM2/HIF-1 positive feedback loop on glycolysis and proliferation of keloid fibroblasts would be investigated. Through this study, we expect to prove PKM2 is a feasible target for anti-keloid therapy.

瘢痕疙瘩的治疗迄今仍是临床难题，其调控机制尚未阐明。近期研究发现瘢痕疙瘩与肿瘤具有相似的有氧糖酵解代谢方式。现已证实某些糖酵解相关因子与肿瘤预后密切相关，成为潜在抗肿瘤靶点，M2型丙酮酸激酶（PKM2）是其中研究热点，其与HIF-1形成正反馈环来调控肿瘤细胞增殖。课题组预实验首次观测到PKM2和HIF-1在瘢痕疙瘩及其成纤维细胞（KF）内显著高表达，且PKM2有激活入核趋势。因此提出假设，PKM2/HIF-1正反馈环对瘢痕疙瘩有氧糖酵解具有重要调控作用，并进一步主导其增殖行为。本课题拟采用基因工程技术，改变PKM2和HIF-1在KF内表达水平和活性；应用ChIP-assay、荧光素酶报告基因技术，探究PKM2与HIF-1是否形成正反馈环；结合组学方法和基因补救技术，阐明PKM2/HIF-1正反馈通路对KF功能的调控作用及其分子机制。通过该项研究，探讨PKM2作为瘢痕疙瘩治疗新靶点的可行性。

项目实施以来，本课题应用免疫组化、药物抑制剂及激动剂、慢病毒敲低等技术，对PKM2在人来源的瘢痕疙瘩标本、瘢痕疙瘩来源成纤维细胞以及裸鼠动物模型的作用进行研究，在瘢痕疙瘩研究领域取得了多项创新：1）明确了PKM2在正常皮肤和瘢痕疙瘩之间的差异性表达以及PKM2在瘢痕疙瘩成纤维细胞中的定位研究；应用PKM2抑制剂，明确PKM2对成纤维细胞生物学行为的作用。在PKM2/HIF-1的正反馈环研究受阻后，及时分析数据，分别选择了其他两个PKM2的相互作用蛋白AMPK和P53。2）揭示AMPK（PKM2的互作蛋白）激动剂能干预由缺氧和TGF-β1诱导的人皮肤成纤维细胞表型向瘢痕疙瘩相关表型转化的过程。3）明确p53 （PKM2的另一个互作蛋白）在正常皮肤和瘢痕疙瘩来源的成纤维细胞之间的差异表达以及敲低p53对瘢痕疙瘩成纤维细胞的生物学行为影响，并进一步证实p53能介导其上游HOXA5对瘢痕疙瘩成纤维细胞的细胞凋亡、细胞增殖、迁移的影响。综上所述，本研究为探讨PKM2及相关两个互作蛋白在瘢痕疙瘩形成和发展中的作用机制研究奠定坚实的基础，为后继进一步研究提供重要线索。本项目完成期间培养硕士生2位，均已取得硕士学位。项目资助发表国内杂志发表论文3篇，国际杂志发表研究论文2篇。