组织移植再生血管化机制的新探索：C53活化MAPKs的血管形成效应

"Add a statement on the importance of revascularization in tissue transplantation of plastic sugery for clinical value". Because revascularization are involved in viability of tissue grafts postoperation. It is well known that MAPKs are the canonical signaling pathways associated with oxidative stress and angiogenesis in plastic surgery field. By bioinformation analysis, it is predicted that C53 contained a motif wich includes the D domain of MAPKs. Moreover, C53, a protein highly expressed in endothelial cells(ECs), was recognized that it would activate MAPKs pathways under hypoxia or oxidative stress by scaffold protein contained docking site (D-site). This will result in cellular migration, survival, proliferation, apoptosis, and at last blood cavity formation of ECs by Reactive oxygen species(ROS), a signaling molecular. Our project will confirm:(1) the scaffold protein contained D-site, a novel protein between the C53 and MAPKs, will effects on in cytoplasm and nucleoplasm. (2) to confirm a complex composed of C53, the scaffold protein contained D-site, and MAPKs will be responsible for angiogenesis and vasculogenesis under hypoxia and oxidative stress. Methods: After hypoxia/reoxygen and H2O2 treatment for ECs, repectively, we will find out and demonstrate the scaffold protein contained D-site by using IP, MS. Moreover, we will explore the molecular mechnism of interaction among the components of the complex composed of C53, scaffold protein contained D-site, and MAPKs in cellular signaling pathway by using proteomic methods, gene mutation and transfection, and laser confocal, etc. At last, we will further investigate the biological effects of this protein complex in vitro and in vivo by using overexpression and knockout expression for ECs, and adenovirus with C53 and scaffold protein contained D-site gene treatment for tissue transplantation of animal model experiment. Predicatlly, this study will identify the novel target for revascularization of tissue grafts including skin, adipose, muscles, and complex tissues.

再生血管化是整形外科相关基础研究的一个重要课题，各种组织移植术都涉及缺氧环境下血运重建，但如何促进移植组织再生血管化的难题现在仍然影响临床治疗效果。MAPKs是与移植组织氧应激和血管化过程相关的经典信号通路，已知血管内皮细胞特异高表达C53，生物信息学预测C53含有结合MAPKs的D domain模体，推测在缺氧环境下的ECs细胞核和细胞质中：(1) C53与MAPKs形成蛋白质复合物；（2）可能存在含D-site的支架蛋白。其结果都是活化MAPKs的信号通路，生物学效应则是改变ECs的迁移、增值、凋亡，和促进新生血管形成。本研究拟在前期工作基础之上，运用免疫共沉淀，质谱分析等多种方法，筛选出D-site支架蛋白，并通过基因突变和过表达以及RNAi干扰等技术深入探讨C53调控MAPKs信号通路的分子机制及其促新生血管形成的生物学效应，为提高移植组织成活能力和促进再生血管化提供新的治疗靶点。

研究背景：组织移植血管化是整形外科相关基础研究的一个重要课题，各种组织移植术都涉及缺氧及氧化应激条件下血运重建的问题，但如何促血管化提高移植物成活改善临床治疗效果仍是目前的难题。 MAPKs 是与移植组织氧应激和血管化过程相关的经典信号通路，而血管内皮细胞特异高表达 C53，阐明其C53-MAPKs对血管再生的影响及其作用机制，将有助于提高移植组织成活能力，并为促进再生血管化提供新的治疗靶点。. 主要研究内容：（1）探讨低氧培养人脐静脉内皮细胞（HUVEC）后，细胞内C53蛋白水平的变化及由C53通过p38 MAPK通路介导的血管化能力的一系列改变。（2）探讨低浓度过氧化氢（H2O2）对HUVEC体外血管形成能力的影响及其作用机制。（3）建立裸鼠体内DIVAA基质胶栓实验模型，在体外实验结果的基础上，证实ERK5的体内促血管生成作用，与体外实验结果相互验证。（4）培养与鉴定脂肪间充质干细胞（ASCs），探讨ASCs对HUVEC体外增殖、迁移及管腔形成能力的影响及其分子机制。（5）建立皮肤移植动物模型，从促进血管化、抑制炎症及促进胶原形成等方面探讨ASCs对皮肤移植成活过程的影响。. 重要结果及其科学意义：（1）低氧培养HUVEC可降低其细胞内C53水平，激活p38 MAPK通路，促进VEGF合成和分泌，促进血管再生。我们首次提出C53可抑制p38 MAPK，C53作为一种近年来新发现的蛋白，有望在后续研究中得到进一步探索，为促进组织移植、伤口愈合中的血管再生，或抑制肿瘤细胞的血管形成过程发挥作用。（2）低浓度H2O2刺激HUVEC体外血管生成，ERK5 MAPK为介导低浓度H2O2刺激HUVEC体外血管所必需，提示ERK5或可作为改善组织移植后再生血管化的有效治疗靶点，具有临床指导意义。（3）建立裸鼠体内DIVAA基质胶栓实验模型，进一步在体内验证ERK5 MAPK促血管生成作用。（4）发现ASCs可以促进HUVECs体外血管生成，并进一步阐明作用机制。（5）建立了大鼠皮肤移植模型，证实ASCs可改善皮肤移植的成活，并具有抑制炎症作用，为临床改善移植皮片成活率提供了重要理论基础。