PEDF-34及其纳米给药系统在角膜损伤修复中的抗炎作用及分子调控机制研究

The pathological healing of corneal injury is closely related to corneal inflammation, which is an important cause of blindness. In our previous studies, PEDF-34 and PEDF-34-nanoparticle-mediated drug delivery had been prepared successfully, and proved active as well. We found that PEDF-34-NPs may inhibit the corneal inflammation by blocking NF-κB activation. This project is a follow-up study. Through in-vitro and in-vivo experiments of H2O2-injured cell (HCFs or PMNs or HUVECs), co-culture system of HCFs and PMNs, and rat corneal alkali-burn model,the dynamic expression of the inflammation factors in the TLR/NF-κB pathway will be detected from cell, protein and mRNA levels, using ELISA, RT-PCR, FCM, fluorescein angiography and cornea flat mounts, histologic and immunohistochemical analysis, for the purpose of more systematic understanding of the anti-inflammatory effects of PEDF-34 or PEDF-34-NPs on corneal injury. In the in-vivo experiment of PEDF-34-NPs, PEDF-34-nanoparticle-mediated drug delivery is expected to be effective, as a stable and continuous way, in the pathological healing of corneal injury. The prospective results will provide theoretical basis for the clinical application of PEDF-34 in the treatment of corneal injury.

角膜损伤的病理性愈合是致盲的重要原因，与炎症密切相关。我们已制备PEDF的功能片段- - PEDF-34及其纳米给药系统（PEDF-34-NPs）并确定活性。前期研究发现PEDF-34-NPs可能抑制NF-κB活化从而减轻角膜炎症反应。本项目是后续研究。通过被H2O2损伤的细胞（HCFs、PMNs及HUVECs）、细胞共培养体系以及角膜碱烧伤动物模型三个层面实验，拟用ELISA、RT-PCR、FCM、荧光血管造影铺片、免疫组化等技术，从细胞、蛋白和mRNA水平进行TLR/NF-κB信号通路各致炎因子动态表达研究，更系统地探讨PEDF-34 在角膜损伤修复中的抗炎作用及其作用机制。借助不同浓度PEDF-34-NPs体内应用，观察稳定而持续应用PEDF-34对角膜损伤修复的影响以及量效关系，论证纳米给药系统是PEDF-34的有效载体。为应用PEDF-34治疗角膜损伤、减少病理性愈合提供理论依据。

角膜损伤的病理性愈合是致盲的重要原因，与炎症密切相关。我们已制备PEDF的功能片段- - PEDF-34及其纳米给药系统（PEDF-34-NPs）并确定活性。通过H2O2损伤的细胞（HCFs和HUVECs）以及角膜碱烧伤动物模型，用Western、RT-PCR、荧光血管造影铺片、免疫组化等技术，从细胞、蛋白和mRNA水平进行TLR/NF-κB信号通路各致炎因子动态表达研究，证实H2O2损伤的细胞（HCFs和HUVECs）以及碱烧伤角膜TLR/NF-κB信号通路各致炎因子从第3天~14天高表达，而PEDF-34-NPs应用后，炎症因子得到有效控制且与剂量正相关。在细胞研究中1.0ug/mlPEDF-34-NPs效价比最高，在大鼠研究中1.0ug/100ulPEDF-34-NPs效价比最高。PEDF-34-NPs能有效治疗角膜损伤，减少病理性愈合，提示PEDF-34NPs可制成眼药水治疗角膜损伤性疾病。