IL-17A调控巨噬细胞极化对视网膜新生血管形成的作用机制研究

Retinal neovascularization is the common pathological changes of many blinding eye diseases. Our previous studies are focused on the pathogenesis, drugs and the effects of gene therapies on RNV and proved that macrophages are closely associated with RNV. The current study is proposed to explore the role and mechanisms of polarization and activation of macrophages modulated by IL-17A on the process of RNV. Previously established animal (including rho/VEGF transgenic mice, rho/rtTA-TRE/VEGF double transgenic mice and IL-17A knock-out mice，etc) and cell models mimicking ocular neovascularization diseases will be investigated by means of retinal flatmounts, immunofluorescence staining, RT-PCR and western blot assays, flow cytometry and co-culture of different cells. Furthermore, IL-17A neutralizing antibody and recombinant IL-17A protein will be used to inhibit or promote the function of IL-17A to further clarify the modulation mechanism of IL-17A on Mf polarization and RNV, thus providing fundamental evidence for IL-17A and Mf as potential targets for the treatment of such ocular neovascularization diseases as diabetic retinopathy and proliferative vitreous retinopathy.

视网膜新生血管(RNV)是许多致盲性眼病共有的病理改变。我们前期的工作已对RNV发生机理、药物及基因治疗效果评估等进行了深入研究，并证实巨噬细胞(Mf)与RNV形成密切相关。本项目拟进一步在已建立的多种动物眼NV疾病模型（rho/VEGF转基因、rho/rtTA-TRE/VEGF双重转基因及IL-17A基因敲除小鼠等）和细胞模型中，应用视网膜铺片、免疫荧光染色、RT-PCR、Western blot、流式细胞术和细胞共培养等方法检测Mf极性转变和信号蛋白表达改变；在此基础上，运用IL-17A中和抗体和重组蛋白干预其作用，观察IL-17A对Mf调控后RNV的改变情况，进一步阐明IL-17A对RNV形成的作用机理，及其与视网膜Mf转录激活之间的关系，进而为把IL-17A和Mf作为DR、PVR等NV性疾病的新治疗靶点提供全面而坚实的实验依据。

眼底新生血管（neovascularization,NV）主要包括脉络膜新生血管（choroidal neovascularization，CNV）和视网膜新生血管（retinal neovascularization，RNV），多见于年龄相关性黄斑变性、病理性近视、增殖性糖尿病性视网膜病变、未成熟视网膜病变、以及继发性的NV性青光眼。目前对于眼底NV性疾病的治疗主要为抗血管内皮细胞生长因子(Vascular endothelial growth factor，VEGF) 治疗，但现有的抗VEGF治疗仍然存在着各种各样的局限性。其巨大挑战主要体现在:(1) 判断是否联合治疗比单一治疗更有效。(2) 设计持续有效的给药途径能够有选择、持久的把药物递送到NV最多见的眼后极部。(3) 清除或重建病理性和无功能的血管以缓解这些NV性眼病视网膜的缺氧状况。因此寻找新的治疗靶点，对于防盲治盲工作刻不容缓。本课题组通过运用oxygen-induced retinopathy(OIR)、CNV、rho/ VEGF、tet-opsin VEGF转基因小鼠等多个动物模型，对眼NV与macrophages (Mphi)活化状态的关系进行深入探究，检测并证实了IL-17对缺氧局部Mphi极性的转变具有调控作用，可促进NV的形成。通过对IL-17基因敲除小鼠的研究表明眼Mphi的清除及极性转变，可抑制RNV以及视网膜下新生血管。除此之外，研究团队发现抑制FLT3、NF-KB在Mphi的极性转化和减少RNV方面也有积极作用。另外，课题组研究期间，通过体外细胞实验和动物实验已证实ATN161和Etanercept可减少眼部NV,抑制IL-23、Endocan和BV8可抑制眼NV。以上研究结果为探索眼底NV性疾病的新治疗靶点和治疗途径提供了全面而坚实的实验依据，亦为NV性眼病的治疗研究开拓了新的研究思路。