Anti-CD26Fab-miR-29b-金纳米复合物抑制皮肤创口愈合过程中胶原合成的实验研究

Facial scar significantly impairs the facial morphology and functions of patients. Fibroblast plays a pivotal role in collagen synthesis and scar formation during skin wound healing, and remains the key target for anti-scar therapeutic manipulation. Recent literature has uncovered that the CD26+fibroblast subpopulation is the primary cell type responsible for collagen biosynthesis and secretion during skin wound repair. Our previous studies have revealed that miR-29b can reduce collagen biosynthesis and scar formation during skin wound healing via post-transcriptional inhibition of HSP47 and Col 1α1 expression. AuNP-miR-29b complex delivery can transfer miR-29b efficiently into fibroblast and in turn exert its biological functions. In the present study, based on the rationale of biological target therapy, we attempt to design and construct the anti-CD26Fab-miR-29b-AuNP complex with antibody fragment production, TEM (transmission electron microscope), LSCM (laser scanning confocal microscope) and FACS (fluorescence-based cell sorting) and perform a series of in vitro and in vivo experiments at the cellular, molecular and animal levels to deliver the miR-29b into CD26+ fibroblast and testify the feasibility and efficiency of this nanocomposite complex therapy to reduce scar formation. This novel nanocomposite-based anti-scar strategy might offer scientific proof-of principle evidence to support the targeted therapeutic strategy against skin scar.

颌面部皮肤瘢痕严重影响患者的面部外形和功能。成纤维细胞是皮肤创口愈合过程中胶原合成与瘢痕形成的主要“参与者”，更是瘢痕防治的关键靶细胞。近期文献报道：皮肤中CD26+成纤维细胞亚群主要负责创口愈合过程中的胶原合成与分泌。我们前期研究发现：miR-29b能转录后抑制HSP47/Col 1的表达，减少胶原合成，抑制瘢痕形成；miR-29b-金纳米复合物能将miR-29b高效转运至成纤维细胞内并发挥生物学效应。本课题基于生物靶向治疗策略，拟在细胞、分子和动物水平上采用抗体片段制备、透射电镜、激光共聚焦和流式分选等技术构建靶向CD26+成纤维细胞亚群的anti-CD26Fab-miR-29b-金纳米复合物并验证其抑制胶原合成减轻瘢痕形成的效能，旨在探索anti-CD26Fab-miR-29b-金纳米复合物靶向CD26+成纤维细胞抑制瘢痕形成的可行性及有效性，为研发皮肤瘢痕防治新策略奠定前期基础。

皮肤成纤维细胞是创伤愈合的主要参与者，也是瘢痕防治的治疗靶点。其中CD26是区分成纤维细胞亚群的分子标志物，在调节皮肤成纤维细胞生物学行为和影响皮肤创伤修复中起着重要作用。本研究基于生物靶向治疗策略，通过建立小鼠皮肤烧伤及切除创面愈合模型，采用免疫组化染色、实时RT-PCR和western blot方法检测创面愈合过程中CD26的表达。将正常皮肤成纤维细胞（NFs）和创伤成纤维细胞（WFs）分离，用流式分选将其分为CD26+NFs、CD26-NFs、CD26+WFs和CD26-WFs 4个亚组，比较它们的增殖、迁移和胶原合成能力。同时利用CD26特异性小分子抑制剂西他列汀靶向抑制CD26+成纤维细胞，分别进行体外和体内试验，进一步评估了西他列汀对皮肤成纤维细胞和伤口愈合过程中CD26的药理抑制作用。并构建西他列汀-Fe2O3纳米颗粒复合物，拟在细胞、分子和动物水平上验证其抑制胶原合成减轻瘢痕形成的效能，结果发现在两种模型的皮肤伤口愈合过程中观察到CD26表达增加。从创面分离的CD26+成纤维细胞增殖、迁移和合成胶原的能力明显强于其他成纤维细胞亚群。西他列汀治疗可有效降低CD26的表达，在体外试验中抑制成纤维细胞的增殖、迁移和胶原合成，并减少体内瘢痕的形成，而西他列汀-Fe2O3纳米颗粒复合物则表现出优秀且长效的抗成纤维细胞胶原合成效果。这些发现表明针对特定成纤维细胞亚群的治疗有望更有效地减少皮肤瘢痕的形成。