ATRA-NLC抑制zymosan 诱导的角膜基质胶原降解及相关免疫调节机制研究

The incidence of fungal keratitis(FK) has been increasing but available antifungal methods are beyond the need of clinical treatment. Host-directed immunomodulatory therapies (HDIT)can provide new development for the clinical therapeutic schedule of FK. Previous studies showed that the ligands of nuclear receptors, such as estrogen，progesterone and medroxyprogesterone mediated in large part through interaction with their specific nuclear receptors, can inhibit extracellular collagen degradation of corneal fibroblasts induced by proinflammatory cytokine, leading to corneal inflammatory pathological damage. In this study we propose a new experimental hypothesis: As a lignd of nuclear receptor, all trans retinoic acid loaded nanostructured lipid carriers (ATRA-NLC)can be a potential HDIT drug for the treatment of FK by activating immune defense in the early phase and inhibiting corneal collagen degradation and the immune inflammatory injuries in the late phase induced by zymosan. We plan to examine the effects of ATRA-NLC on the inflammatory factors expression induced by zymosan by culturing rabbit corneal fibroblasts in two dimension and three dimension and by fungal keratitis animal model, and to examine the regulating effects of ATRA-NLC on fungi immunology and corneal collagen degradation induced by zymosan. Since the objective of ATRA-NLC is to initiate or enhance protective antimicrobial immunity in order to limit inflammation-induced tissue injury, the effect of ATRA-NLC on the nuclear receptor and cell signal transduction pathway of the inflammatory fators induced by zymosan is clarified . Also,we plan to observe the role of ATRA-NLC on the corneal collagen degradation induced by zymosan and immunoregulatory strategy for FK. The findings of this study will provide therapeutics host-directed immunoregulatory strategy for FK by the ligands of nuclear receptors in the future.

真菌性角膜炎（FK）发病率升高，真菌耐药难题使FK的药物治疗已不能满足临床需求，针对宿主免疫调节治疗（HDIT）成为FK药物治疗的新方向。核受体在细胞核内调控多种目标基因表达,前期研究发现核受体配体雌激素、黄体激素、醋酸甲孕酮可抑制炎前因子诱导的角膜基质胶原降解过程。结合FK研究进展提出实验假说：核受体配体全反式维甲酸加载的纳米结构脂质载体（ATRA-NLC）可早期激活zymosan诱导的角膜基质细胞及FK家兔的免疫防御反应，抑制zymosan诱导的角膜基质胶原降解及晚期的炎性损伤过程，可成为FK的理想HDIT制剂。通过建立角膜基质细胞二维及三维胶原凝胶培养体系，进行体内实验，探索ATRA-NLC对酵母聚糖刺激炎症因子表达的核受体及细胞信号转导途径，明确其对酵母聚糖诱导的角膜基质胶原降解的作用及其对宿主相关免疫防御反应的调节作用，将核受体作为新的药物靶标，为角膜真菌感染的HDIT提供依据。

主要研究内容：①.体外实验（这部分为重点内容）：构建角膜基质细胞二维培养体系：观察ATRA-NLC对zymosan诱导的角膜基质细胞炎症因子表达的核受体作用途径（基因组）及细胞信号转导途径（非基因组）。.构建角膜基质细胞三维胶原凝胶培养体系：观察zymosan诱导角膜基质胶原降解情况，阐明ATRA-NLC通过调节MMPs/TIMPs产生抑制zymosan诱导的角膜基质胶原降解过程。②.体内实验：建立LPS角膜基质内注射动物模型，探讨ATRA-NLC治疗效果及其对FK获得性免疫反应的调节机制。③.毒理学及细胞增殖实验：进行ATRA-NLC对细胞损伤及细胞增殖能力的影响研究，探讨其细胞毒性及应用潜力。重要结果：①.成功建立了角膜基质细胞二维及三维培养体系，证实ATRA-NLC对zymosan诱导的角膜基质细胞炎症因子表达（IL-17,ICAM-1,MIP-2,mcp-1,Ip-10,IFN-r）有抑制作用，并同时促进IL-4,10的表达，可抑制zymosan依赖的TLR-2表达，证实ATRA的受体作用途径是ATRA-a,r途径而非beta途径，证实其细胞信号转导途径为 NF-KB-p65, P-ERK, P-IκBα信号分子，并证实了其免疫调节作用。②. 制备全反式维甲酸纳米微粒ATRA-NLC用从头乳化法制作。平均粒径200nm,高效液相色谱检测ATRA-NCL,耐光性研究良好。测定ATRA浓度曲线方程，物理稳定性良好，在4°条件下存放一个月以上无明显沉淀和结晶，高速离心2小时未见固体析出。不耐高温。加热至120°以上时明显颜色变黑，继续加热凝固成黑色膏状物。③.ARTA可抑制MMPs表达，并可抑制LPS诱导的角膜溃疡形成。④.ARTA没有细胞毒性，对角膜基质细胞增殖能力没有影响。 科学意义：通过上述实验过程，我们证实ATRA具有抑制角膜基质细胞炎症反应，这一抑制作用通过对上述细胞因子趋化因子，MMPs产生的作用及信号转导分子的作用来实现，ATRA具备成为临床上真菌性角膜炎的辅助制剂的临床应用潜力。