自噬相关基因Atg7调控血管内皮细胞参与创面血管新生的实验研究

Angiogenesis is a biological process of forming new blood vessels from existing vessels, which is critical for wound healing and tissue repair. The first step in new vessel formation is the binding of growth factors to their receptors on the endothelial cells of existing vessels. The activated endothelial cells are now able to proliferate and migrate into the wound, a process also known as 'sprouting'. The newly built sprouts form small tubular canals interconnect to others forming a vessel loop. Thereafter, the new vessels differentiate into arteries and venules and mature by a further stabilization of their vessel wall via the recruitment of pericytes and smooth muscle cells. Finally, the initial blood flow completes the angiogenic process. Howover, the mechanisms involved in this process have not been fully understood. Autophagy serves as a dynamic process of subcellular degradation, which is found to be important for angiogenesis and tissue repair. Autophagy occurs by environmental stress, such as hypoxia and injury. Induction of autophagy could promote angiogenesis. Recently we have found that the conditional knockdown of autophagy-related gene 7 impaired angiogenesis of granulation tissue, and inhibited wound healing. According to above-mentioned findings, we hypothesise that Atg 7 might be participate in wound repair possiblely by influencing wound healing. In the present study, endothelial cells serves as an important traget. We will develop a mose model of full-thickness cutaneous wound healing, and observe the effect of the different condition knockdown of Atg 7 on wound healing and new vessel forming, and explore their relationship. We will also develop an angiogenesis model by implanting matrigel into the back of mice, and examine the effect of different conditional knockdown of Atg 7 on angiogenesis in the model. To investigate the possible mechanism by which Atg 7 influences angiogenesis, the endothelial cells will be cultured, the effect of the disfunction of Atg 7 on the proliferation, migration and sprout will be observed. Based on above-designed experiment, we will try to reveal the role of autophagy in wound-related angiogenesis, and clarify the possible mechanisms by which autophagy participates in wound healing through regulating angiogenesis in wounded sites. This study will be helpful to understanding the cellular and molecular mechanisms of wound healing and angiogenesis, as well as improving the outcome of wound repair.

血管新生是创面愈合过程中的重要环节和组织修复的基础，血管新生起自内皮细胞的增殖和迁移，出芽是新生的标志，然而创面愈合过程中内皮细胞参与创面血管新生的调控机制尚未阐明。既往研究表明：自噬一种动态的亚细胞结构清除方式与血管新生和组织修复密切相关，损伤是激活自噬的一种方式，诱导自噬可促进血管新生。我们最近的研究发现：在自噬相关基因Atg7条件型基因敲除杂合子小鼠，创面肉芽组织的血管新生受损，创面愈合减慢。据此推测:Atg7可能通过调控血管新生参与创面修复。本立项以血管内皮细胞为主要研究对象，通过建立Atg7条件型基因敲除小鼠创面模型，利用细胞培养，"失能"Atg7功能等手段，观察自噬抑制后内皮细胞增殖、迁移和出芽的变化，血管新生的改变，及其与创面愈合之间的关系，探讨Atg7在内皮细胞参与创面血管新生中的作用和机制，试图阐明自噬调控皮肤损伤修复的机制，为改善组织修复结局，提高修复质量提供理论基础。

血管新生是创面愈合过程中的重要环节和组织修复的基础，，然而创面愈合过程中内皮细胞参与创面血管新生的调控机制尚未阐明。我们最近的研究发现：在自噬相关基因Atg7条件型基因敲除杂合子小鼠，创面肉芽组织的血管新生受损，创面愈合减慢。据此推测:Atg7可能通过调控血管新生参与创面修复。本立项以血管内皮细胞为主要研究对象，通过建立Atg7条件型基因敲除小鼠创面模型，利用细胞培养，“失能”Atg7功能等手段，观察自噬抑制后内皮细胞增殖、迁移和出芽的变化，血管新生的改变，及其与创面愈合之间的关系，探讨Atg7在内皮细胞参与创面血管新生中的作用和机制，试图阐明自噬调控皮肤损伤修复的机制，为改善组织修复结局，提高修复质量提供理论基础。.Atg7内皮细胞特异性基因敲出小鼠并不产生内皮细胞丰富器官明显的组织结构的改变，和Atg7F/F对照鼠相比较，Atg7EC-/-小鼠创面上皮化明显加快，通过敲出Atg7导致内皮细胞自噬损害并不影响内皮细胞增殖和血管再生。Atg7F/F对照鼠相比较，Atg7EC-/-小鼠创床内炎性细胞如巨噬细胞、T淋巴细胞侵润增加，Atg7EC-/-小鼠募集炎症细胞的能力增强。鉴于我们的研究中发现：通过敲出Atg7导致内皮细胞自噬损害并不影响内皮细胞增殖，迁移和创面床的血管再生，然而却促进了创面的愈合。细胞的分泌功能影响对创面愈合过程中的重要细胞成分成纤维细胞和上皮化过程中起重要作用的表皮干细胞功能。我们发现：自噬受损组成纤维细胞和表皮细胞增殖能力增强和迁移明显加快。我们发现自噬功能的抑制导致内皮细胞分泌功能的增强，我们已取得的发现为阐明条件型Atg7基因剔出影响创面愈合的分子机制提供新的线索。自噬在糖尿病、糖尿病并发症以及创面愈合过程中均发挥重要作用。为此我们建立了糖尿病模型，验证微血管病变情况下，自噬-内皮分泌-创面愈合的关系。我们发现：自噬的基因敲出并没有影响糖尿病小鼠的创面愈合，提示糖尿病条件下自噬功能已经异常。我们的研究结果提示糖尿病导致自噬功能异常是糖尿病血管内皮功能障碍的原因之一。