支架中昆布多糖对巨噬细胞的免疫调控活性及其用于慢性难愈皮肤缺损修复的研究

The scaffold with functional component of active immunomodulatory on immune cells, and for designing tissue regeneration scaffolds with immunomodulatory functions，which promotes proliferation and differentiation of adult cells and synthsis of extracelluar matrix, are one of the hot spots of biomaterials. In this project, laminarin, a kind of seaweed polysaccharide with anti-infection activity as we had disclosed in a former NNSFC, is designed to stimulate immunocytes to produce specific cytokines (such as IL6,IL-10) for promoting proliferation and ECM synthesis of fibroblasts with fibroblasts as model cells, and co-culture of raw264.7/fibroblasts, and other celluar and molecular biology methods. After the relationship of immunomodulation of laminarin polysaccharide and its derivatives with different molecular structures with the expressions of cytokines and growth factors, will be evaluated and disclosed with RAW264.7, mouse peritoneal macrophage,208F mouse fibroblasts, the fibrous scaffold with collagen I will be designed by co-electronspunning laminarin or its modifications and collagen I with adjusting components and electronspun time. Also, the favorable co-electronspunning membrane was chosen by testing the fibrous membrane stimulating immune cells expressing cytokines which upregulates adult cells expressing ECM and tissue repair. Skins of normal and diabetic mice II as organ models, are designed to test the immunomodulated membrane to accelerate the skin regeneration. This study is expected to clarify the structure-activity relationship between immunomodulation of materials and tissue repair acceleration, find a new way to the tissue regeneration scaffold research, and provide new materials and new methods for the rapid regeneration of skin tissue.

通过支架材料中功能组分对免疫细胞活性的“主动”调控，促进体细胞（种子）增殖和胞外基质合成，达到快速修复缺损的目标，是组织工程和再生医学未来的研究热点之一。在前期探明海藻多糖-昆布多糖抗感染活性的基础上，本项目利用它具有刺激免疫细胞产生特定细胞因子的特性，以成纤维细胞为体细胞模型，通过免疫细胞/体细胞共培养、细胞分子生物学等手段弄清昆布多糖调控免疫细胞释放特定因子（如IL6，IL-10等），促进成纤维细胞的快速增殖和胞外基质合成机制和构效关系，并设计和电纺出含昆布多糖免疫活性支架。以修复糖尿病大鼠皮肤溃疡组织为模型，检验所设计功能支架对慢性难愈皮肤缺损的修复能力，为最终获得一类具有免疫调控活性支架奠定基础。该研究弄清昆布多糖“主动”调控免疫细胞活性的构效关系，不仅具有重大科学意义,还为寻找新型功能再生支架开辟新途径，同时也为困扰临床上缺损组织快速修复问题的解决提供新技术，预期有广阔应用前景。

寻找具有免疫调控活性的材料是生物材料领域研究的热点之一，本项目拟通过昆布多糖改构调控其免疫活性，设计成敷料用于难愈性创面的修复。（1）通过降解、乙酰酯化、胺化等改构方式，得到不同结构的葡聚糖。以巨噬细胞（RAW264.7）为模型，测定改构葡聚糖的免疫调控活性(关键是表达 IL-1, IL-6, IL-10,TNF-α, TGF-β量)；确定材料含量与细胞因子表达量关系，发现酯化葡聚糖分子量约2.3万Da，浓度为500ng/ml就明显刺激免疫细胞表达细胞因子，因此可调控葡聚糖的结构使其在后续添加到支架中维持其刺激活性。（2）酯化葡聚糖本身，以及与I型胶原能方便电混纺、或同轴纺丝，可制备浓度梯度、双面亲疏水性纤维状支架或复合支架，可满足皮肤修复的需要。与大鼠成纤维细胞、RAW264.7细胞单独培养或构建共培养体系，研究发现细胞增殖良好，在支架中分布均匀；同时共培养体系发现在含酯化葡聚糖的复合支架中，成纤维细胞的增殖速度明显快于纯胶原组；通过定量检测IL-10、TGF-BETA的表达量、合成胶原(I，III型胶原)量，发现当酯化葡聚糖/胶原质量比为1:1时，这些促组织修复的细胞因子最高，且在3-7天达到极值，这表明该组分有助于创面的快速修复。（3）通过诱导大鼠糖尿病，建立了创面难愈模型，发现普通敷料（胶原等）修复这些创面，比正常鼠创面修复时间慢3-7天（总2-3周）。采用酯化葡聚糖/胶原质量比为1:1的混纺纤维膜作为敷料，修复时间明显缩短，组织学分析表明经优化的支架明显促进基质的合成，从而达到对难愈性创面的快速修复。总之，通过研究发现酯化葡聚糖/胶原按1:1配比混纺得到的复合纤维支架有明显刺激免疫细胞合成有利于组织修复的细胞因子，促进成纤维细胞合成胞外基质，加快难愈性创面的修复。