新型类弹性蛋白融合 bFGF 功能材料的制备及皮肤缺损修复研究

The basic fibroblast growth factors (bFGF) play critical roles in wound healing and tissue regeneration through recruiting neutrophils, monocytes, macrophages and fibroblasts to injured part via chemotactic effect. Recombinant bFGF has been clinically applied to cure bedsore, incision, burns, chronic ulcer and brain injury caused from ischemic central nerve damage. However, bFGF need a long-time unremitting interaction with its receptor to exert its function, during which bFGF is prone to be degraded by protease and the time of drug action is reduced. For the clinical application in wound healing, the pressing problem is to develop the stability of bFGF to prolong its action time. .Elastin-like polypeptides (ELPs) is genetically derived from the sequence bio-inspired by which found in natural elastin. The most widely used motif has the sequence (VPGXG)n, where the guest residue X is any amino acid except proline and n symbolizes the number of pentapeptide repeats in the ELPs, although additional motifs such as VPG, VPGG, GVGVP, IPGVG or VAPGVG have also been explored in natural elastin. ELPs are highly biocompatible and exhibit a huge potential for biomaterials development. Among them, the most striking feature is that of a thermally responsive reversible phase transition. These characteristics make ELPs attractive for drug delivery, appealing as materials for tissue repair or engineering, and improve the efficiency with which recombinant proteins can be purified easily. ELP fusion proteins (referred to as ELPylation) inherit the reversible phase transition property. Further, the temperature threshold of ELPs alters along with the change of concentration, pH, ion, amino acid sequence, and the chain length of peptide. .The aim of this proposed project is to prolong the action time of bFGF through constructing fusion protein of bFGF and ELPs with different amino acid sequence and chain length. Firstly, a series of special expression vector, in which the bFGF sequence is recombined to the carboxyl/NH3 terminus of ELPs, will be established and used to transform the competent cells of Escherichia coli BL21 (DE3) to stably over-express the fusion peptide termed ELPs-bFGF. The biological activity of the series of ELPs-bFGF with different amino acid sequence and molecule length will be initially analyzed through cell proliferation and adhesion assay. Second, the recombinant ELPs-bFGF, with bFGF function and a phase transition of 37℃ or approximate 37℃, will be chosen to prepare kinds of hydrogels to deliberately analyze the proliferation and adhesion of fibrablasts cultured in ELPs-bFGF hydrogels using sandwich ELISA and fluorescent histochemistry. Finally, the hydrogels of ELPs-bFGF will be employed to repair the full-thickness defect of mouse. The biofunction and the stability of ELP-bFGF will be comprehensively evaluated on the basis of sandwich ELISA, western blot, fluorescent histochemistry and laser confocal microscope. The applicant and its group member are confident that the results will benefit the clinical application of bFGF.

碱性成纤维细胞生长因子bFGF在组织损伤修复和伤口愈合中具有重要作用，重组bFGF已被用于多种创面的临床治疗。但是，bFGF容易被酶降解，活性半衰期短，严重影响了bFGF的应用效果。类弹性蛋白ELPs具有温度敏感相转变特性，当温度高于其相变温度时自聚集成水凝胶，在药物靶向与缓释、组织修复及构建功能型ELPs融合蛋白方面备受关注。课题组拟利用基因工程技术将bFGF与氨基酸组成和肽链长度不同的ELPs构建成一系列融合蛋白ELPs-bFGF；通过细胞增殖、迁移和粘附实验，筛选出兼具bFGF生物学活性且相转变温度接近37℃的ELPs-bFGF并制备其水凝胶；利用形态学观察、夹心ELISA、蛋白质印迹、免疫荧光染色、激光共聚焦显微镜等技术分析ELPs-bFGF在细胞水平和在体皮肤缺损修复中的生物学活性与实际作用效果。获得bFGF缓慢释放的高活性ELPs-bFGF，为皮肤损伤修复提供一种新的功能材料。

碱性成纤维细胞生长因子（bFGF）是由 146 个氨基酸残基构成的碱性多肽， 可以通过趋化作用使中性粒细胞、单核细胞、巨噬细胞、成纤维细胞等向损伤部 位聚集，促进血管、神经和肉芽组织生长，加速组织损伤修复与伤口愈合。重组 bFGF 已被用于多种创面的临床治疗。但是，bFGF 在体作用时易被酶降解，活性 半衰期短，严重影响了 bFGF 的功能发挥。项目组利用基因重组技术先后设计构建了40种重复基序略有不同的ELPs-bFGF，在大肠杆菌中进行过表达、分离纯化、细胞实验、水凝胶包埋实验、小鼠皮肤缺损修复实验，筛选获得2种同时具有ELPs的相转变特性及bFGF活性的融合蛋白ELPs-bFGF1和ELPs-bFGF7；项目组还按照研究计划建立 ELPs-bFGF 水凝胶制备方法，含有β-甘油磷酸钠的温敏型壳聚糖水凝胶适合负载bFGF或ELPs-bFGFs，包埋在水凝胶中的小鼠成纤维细胞L929可在bFGF或ELPs-bFGFs刺激下加速增殖，增加创伤修复因子VEGF、KGF、TGFβ等的合成；小鼠全皮缺损修复实验结果表明，负载有ELPs-bFGF1和ELPs-bFGF7壳聚糖水凝胶能够通过增加部分损伤修复因子的表达促进损伤修复，新生皮肤厚度接近正常。

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