IL-10基因修饰人羊膜间充质干细胞局部调控巨噬细胞促进创面再生愈合的研究

Fine adjustment of inflammation and regeneration are regarded as a core in regenerative healing. It is a key of regenerative healing on no scar in fetal, which inflammatory response and immunologic balance is mediated by IL-10. Wound macrophage is a key cell of inflammatory response. IL-10 and bone marrow mesenchymal stem cell can control by regulation of macrophage phenotype and function，then to promote wound repair. But it is difficulty to realize physiological regeneration healing. Human amniotic mesenchymal stem cells have antiflammatory, immunoregulation and paracrine function and so on. We will decorate hAMSCs as a IL-10 gene expressing victor. These technologies will be used such as flow cytometry, immunofluorescence and immunohistochemical staining, co-culture, ELISA methods, real-time PCR, Western blot and small animal in vivo imaging. The effects will investigate that continue IL-10 high expressing cooperate with hAMSCs activity treat with skin wound on regulation of macrophage phenotype and function, inflammation and immune，regeneration healing. The therapeutic effect of IL-10 gene modificatory hAMSCs will be clarified on wound healing, while the change of macrophage phenotype and function, and regulation of inflammatory and immue will be explained. The about results will apply some important basis and new target on promoting the regeneration of healing of skin.

创面炎症与再生的精细调控被认为是再生愈合的核心，IL-10介导的炎症反应与免疫平衡是胎儿无瘢痕再生愈合的关键,创面巨噬细胞是创面炎症反应的关键细胞，IL-10、骨髓间充质干细胞（MSCs）均调控巨噬细胞表型与功能而控制炎症，促进创面修复，但难以实现生理再生愈合。人羊膜间充质干细胞（hAMSCs）具有抗炎、免疫调节和旁分泌等功能。本研究将以hAMSCs为载体进行IL-10基因修饰，运用流式细胞术、免疫荧光和免疫组化、共培养、ELISA法、定量PCR、蛋白印记、小动物活体成像等技术，观察创面局部持续高表达IL-10，协同hAMSCs的功能活性，对创面巨噬细胞表型与功能调控、炎症与免疫及皮肤创面再生愈合的影响，明确IL-10基因修饰hAMSCS对创面修复治疗效果的同时，be巨噬细胞表型与功能及有关炎症免疫调控的变化，为以MSCs为载体的基因治疗用于皮肤损伤，促进再生愈合提供重要依据和新的靶标。

无瘢痕愈合一直是创伤修复追求的理想目标。胎儿无瘢痕愈合的机制研究较多，再生愈合与伤口炎症反应有关，因此如何调控创面修复过程中伤口炎症的精细平衡值得探讨。巨噬细胞是出生后创面免疫应答和炎症调控的关键细胞，人羊膜间充质干细胞（hAMSCs）具有抗炎、免疫调节和旁分泌等功能，已有研究证明胎儿无瘢痕愈合的炎症抑制主要是由IL-10介导，可推测经过IL-10基因修饰的人羊膜间充质干细胞可在一定程度上调控巨噬细胞的表型变化从而促进创面的再生愈合。本课题分离培养鉴定了hAMSCs、巨噬细胞、成纤维细胞，通过重组IL-10慢病毒表达载体感染hAMSCs后完成了hAMSCs及巨噬细胞共培养，以及它们的上清液与成纤维细胞共培养，并进行了生物学特性检测；制备C57BL/6小鼠皮肤创伤模型，通过炎症反应评分，组织病理学检查、CM-Dil示踪、冰冻切片免疫荧光双染色、PCR观察IL-10基因修饰hAMSCs局部多点移植对炎症与修复的影响；IL-10基因敲除杂合子小鼠繁殖后PCR鉴定基因型。结果显示：IL-10-hAMSCs细胞能够有效促进巨噬细胞表型转化并且效率较高，上清液中IL-10转染组IL-12表达随着共培养时间延长含量逐渐降低更为明显，IL-10-hAMSCs组成纤维细胞增殖速率明显较快，IL-10转染组TGF-β、I型胶原、III型胶原mRNA相对表达量更高。初步结论：IL-10-hAMSCs能够通过影响巨噬细胞表型与功能，控创面炎症反应，同时可调节成纤维细胞功能，影响修复相关因子表达，促进创面再生愈合及抑制纤维化。