碱性成纤维细胞生长因子糖化后受体静默的机制研究

Under the condition of diabetes mellitus, chronic hyperglycemia may easily induce the form of advanced glycated end products (AGEs), and thus roles their morbific effects to impaired wound healing. Some researches, as well as our previous study, have shown that, basic fibroblast growth factor (bFGF) could be glycated in case of high glucose. The glycated bFGF may probably lead to difficult healing by influencing the biological behavior of endothelia cells. Other research indicated that artificially glycated bFGF expressed AGE and was not able to bind to receptors of either FGF (FGFR) or AGEs (RAGE). Dependingly, we inferred that, the tertiary structure of bFGF had been changed out of the presence of molecule with AGEs after glycation. As the result, the binding domain to the receptors was wrapped, leading to the phenomenon named receptor silencing. If receptor silencing was there, glycated bFGF may not role its morbific effects in receptor-involved pathway as it was considerred. To varify the existence of receptor silencing and its possible mechanism, we are going to refine the method of preparing glycated bFGF, and detect its binding affinity to FGFR or RAGE. The tertiary structure of glycated bFGF will be also inspected. As such, the knowledge of receptor silencing after bFGF glycation could be initiated. Meanwhile, by knowing the expression of critical molecule in the pathway with the receptors, the secondary evidence will be presented to prove receptor silencing.

糖尿病状态下长期高糖环境易诱发糖化终末产物AGEs的生成，介导多种导致创面难愈的病理效应。多项研究和我们的前期实验发现，生长因子bFGF在高糖环境下可以被糖化，影响血管内皮细胞的生物学行为，并可能导致创面难愈。另外，人工制备糖化bFGF可检测出携带AGEs基团，且丧失与bFGF受体FGFR、AGEs受体RAGE结合的能力。由此我们推测，bFGF糖化后，因蛋白质三级结构可能发生变化，结合区域被屏蔽，导致无法与相应受体结合，产生受体静默现象。而这一机制的存在，提示糖化bFGF的病理效应并不通过受体途径。为证实bFGF糖化后的受体静默现象及其机制，我们拟通过改良糖化bFGF制备方案，检测糖化bFGF与FGFR、RAGE两类受体的结合状态，研究bFGF糖化后蛋白质空间构象的改变，初步阐明bFGF糖化后受体静默的发生机制。同时，通过检测受体相关的信号通路关键分子表达，间接提供受体静默现象存在的依据。

糖尿病难愈创面的发生是一个多因素、多环节参与的病理过程，碱性成纤维细胞生长因子bFGF是创面愈合的重要因子，多种研究提示，糖尿病皮肤组织中bFGF可能发生功能上的变化，特别是糖化bFGF的生成，使创面未能发挥正常的促愈效应，从而表现出损害修复过程的综合效应。本项目拟通过研究糖尿病皮肤组织bFGF是否存在高水平糖化现象，以及bFGF和糖化bFGF对人脐静脉内皮细胞HUVEC的效应，围绕bFGF糖化后的空间构象，证实生长因子糖化后受体静默机制，为糖尿病难愈创面治疗中的生长因子干预和糖化效应的后续阻断策略提供可靠的思路。鉴于bFGF的经济效益以及自身化学性质，并不能获取足够大的量去开展部分研究，受限于商品化bFGF本身化学结构的不稳定性及目前并无bFGF现存核磁图谱，我们暂时无法测绘糖化bFGF的三维结构图谱。.就目前研究结果显示：糖尿病组及非糖尿病组皮肤组织中均可检测到糖化bFGF表达，糖尿病组皮肤组织的糖化bFGF表达较高。质谱显示bFGF相对分子量为16407Da，糖化bFGF分别为16407Da、16569Da、16731Da、16893Da。高效液相色谱法显示，糖化bFGF同H2O和bFGF相比，糖化bFGF在6-7min出现差异色谱峰，随样品浓度增加，色谱峰呈增高趋势。SPR显示bFGF、糖化bFGF结合FGFR、RAGE呈浓度依赖性。bFGF、糖化bFGF与FGFR、RAGE结合的平衡离解常数分别属同一级别。与正常培养的HUVEC相比，bFGF干预后第1、3、7天，细胞增殖百分数显著增强；糖化bFGF干预后第1天细胞增殖百分数显著增强，第3、7天未见明显差异；bFGF明显促进HUVEC血管形成，糖化bFGF明显抑制血管形成；干预后第7天，糖化bFGF组与bFGF组相比，细胞凋亡百分数显著增加，而第1、3天无明显差异。.因此，我们得出以下结论：1，糖尿病人体皮肤组织中存在bFGF高水平糖化现象。2，糖化bFGF模型制备成功。bFGF与糖化bFGF均可结合FGFR和RAGE。3，糖化bFGF干预后，HUVEC血管形成效应受到抑制。