多光子荧光寿命显微成像以及新型诊疗方案在银屑病应用中的研究

Psoriasis is a chronic, inflammatory skin disorder affecting 2–3% of the population. As a multisystem disease, psoriasis is also known to affect the joints in 6–42% of patients, and more recent studies have uncovered an increased risk of myocardial infarction and stroke, possibly due to chronic elevation of inflammatory cytokines. Current therapies for psoriasis include topical agents, oral systemics, and biologic agents. To minimize the side effects of these therapies, molecular-targeted cancer therapies, including monoclonal antibodies therapy, have been developed with limited success. In this study, we will being developed a new type of molecular-targeted psoriasis therapy, photoimmunotherapy (PIT), that uses a target-specific photosensitizer based on a near-infrared (NIR) fluorescence dye, ICG-sOSu, conjugated to biologic agents (Infliximab,Ib) targeting TNF-α, as theranostic agents (ICG-sOSu-Ib). Due to the intense research focusing on the treatment and the cure of this chronic disease it is fundamental to have the means of monitoring the effect of various treatments from the macroscopic down to the microscopic level of organization of skin. We will be using multiphoton fluorescence lifetime imaging microscopy (MP-FLIM) to demonstrate the feasibility of an in vivo imaging approach for a psoriasis model on rat skin, which has the potential to supplement the noninvasive clinical diagnosis of psoriasis. In addition, we will also be investigating changes redox state of the epidermis, studying on pharmacokinetic of ICG-sOSu-Ib in psoriatic lesions, observing varieties of immune cells and cutaneous immune responses during the PIT treatment. MP-FLIM can be used as a promising molecular imaging tool for diagnosing psoriasis and monitoring the treatment outcome in preclinical models and potentially in patients.

银屑病是一种慢性炎症皮肤病，患者占世界人口的2-3%，6-42%的患者出现关节性障碍。研究发现：随慢性炎症细胞因子的增多，银屑病可引发心肌梗塞和中风等并发症。当前治疗银屑病的方法主要包括局部外用，口服和生物制剂。为将现有疗法的副作用降到最低，已提出了分子靶向疗法，其中包括单克隆抗体，但疗效不理想。本项目首次提出了一种新型的银屑病治疗方法：光免疫治疗，即近红外(NIR)荧光染料ICG-sOSu与TNF-α的英夫利昔单抗共轭，作为一种新型诊疗剂(ICG-sOSu-Ib)，经光照后进行治疗。同时，本项目还将利用多光子荧光寿命显微成像（MP-FLIM）建立临床无损诊断银屑病的新方法，分析皮肤表皮氧化还原状态，ICG-sOSu-Ib在皮肤内药代动力学以及光免疫治疗后皮肤的免疫反应。MP-FLIM作为分子水平成像技术有望在临床诊断银屑病并评估治疗中发挥重要作用。

荧光寿命不受探针浓度、激发光强度和光漂白效应等因素影响，荧光寿命显微成像技术（fluorescence lifetime imaging microscopy，FLIM）在监测微环境变化、反映分子间相互作用方面具有高特异性、高灵敏度、可定量测量等优点，近年来已被广泛应用于生物医学等领域。..本项目基本实现了所提出的银屑病分子靶向治疗方案并改进为光热治疗结合免疫治疗。项目仍选择近红外(NIR)荧光染料ICG-PEG5K与针对银屑病TNF-α的英夫利昔单抗结合，成功制备了新型诊疗剂(ICG-PEG5K-Ib)。项目利用荧光寿命系统（单双光子）和非介入式多光子光活检层析成像系统Multiphoton Tomograph MPTflex(Jenlab)成功建立了诊断、评估BALB/c 白鼠背部皮肤银屑病皮肤的新方法(Phasor-FLIM)；但项目所依托FLIM平台不能观察到皮下血管，因此调整为分析银屑病皮肤棘层上皮细胞新陈代谢状态(NADH和FAD)，并提出利用Anti-TNF-alpha/PE-Cy7分析诊疗剂(ICG-PEG5K-Ib在银屑病皮肤内的药代动力学以及治疗前后(ICG-PEG5K-Ib结合808nm激光光照)皮肤的免疫反应。我们认为FLIM技术有望在临床诊断银屑病并监测治疗中发挥重要作用。