新型多肽HM-3治疗脉络膜新生血管的研究

发布时间：2018-02-26 09:15

本文关键词： 脉络膜新生血管 HM-3 整合素信号通路代谢动力学　出处：《南京医科大学》2014年博士论文　论文类型：学位论文

【摘要】：目的：年龄相关性黄斑变性(Age-related macular degeneration, AMD)是西方发达国家首位的致盲眼病,而脉络膜新生血管(choroidal neovascularization,CNV)是导致湿性AMD患者严重和不可逆的视力损伤的最主要原因。近年来,我国AMD的发病率逐年升高。目前AMD的病理机制仍不明确,但是越来越多的研究显示血管内皮生长因子系统及整合素信号通路参与CNV的发生和发展,包括细胞外调节蛋白激酶(extracellular regulated protein kinases, ERK)和磷酸化的ERK在内的多种信号分子,这些信号途径构成的网络,在CNV的病理过程中起着重要的作用。HM-3药物是由中国药科大学自主研发的一种抗新生血管多肽,不仅具有内皮抑素的活性片段,更有能与整合素特异性结合的RGD序列,不仅可以通过内皮抑素的作用抑制新生血管,还可参与整合素信号通路,并通过其起到抑制新生血管的作用。前期的体外实验研究也已经显示HM-3具有抗炎,抗氧化,抗肿瘤,抗血管新生等多种功效。本项研究通过与中国药科大学合作,主要探讨HM-3药物玻璃体腔给药对治疗CNV的作用及可能机制,此外,也对玻璃体腔给予HM-3药物在小鼠眼内及全身的代谢动力学进行分析,为HM-3药物开发成为治疗CNV的新型多靶点药物提供基础。方法：本项研究分两部分进行。第一部分,我们采用激光诱导C57BL/6小鼠产生CNV模型,造模后,治疗组经玻璃体注射10,20,40mg/kg/HM-3药物,阴性对照组给予注射生理盐水,阳性对照组给予玻璃体腔注射Avastin药物。在激光造模后的第7天,采用荧光标记的葡聚糖血管灌注,测量RPE-脉络膜铺片上CNV的面积；通过荧光血管造影来评价CNV的渗漏；造模后第7天,采用组织学来评估激光损伤部位新生血管的浸润；同时分别通过荧光定量PCR方法检测血管内皮生长因子(vascularendothelialgrowthfactor, VEGF),肿瘤坏死因子-α(tumor necrosis factor, TNF)在视网膜色素上皮-脉络膜复合体的表达；酶联免疫吸附测定法(Enzyme-Linked ImmunoSorbent Assay, ELISA)检测视网膜色素上皮-脉络膜复合体中VEGF和TNF-a的表达水平。通过Western blotting,检测视网膜色素上皮—脉络膜复合体中ERK1/2及p-ERK1/2的表达。第二部分,我们采用玻璃体腔注射不同浓度的HM-3药物,浓度分别为10,20,40mg/kg,选取12个时间点分别为分别为3,6,12,24,48,72,96,120,144,168,192和216h,处死小鼠后,摘除眼球并留取血液备用(肝素抗凝),分离出完整的视网膜和RPE-脉络膜组织层,采用间接竞争ELISA法分别检测在不同时间三种不同组织中的药物代谢参数。结果：第一部分,与生理盐水对照组相比,HM-3治疗组CNV面积显着减少(P0.05)、CNV渗漏也显著减少(P0.001),且20mg/kgHM-3药物组与阳性对照Avastin组结果相似。HM-3治疗能显著抑制激光损伤部位新生血管的浸润(P0.05)。激光损伤的脉络膜中,VEGF的表达可以被HM-3所抑制(P0.05),此外HM-3能抑制RPE-脉络膜中TNF-a(P0.05)的表达,同时激活激光损伤早期色素上皮-脉络膜复合体中ERKl/2和p-ERK1/2信号途径(p0.05)。第二部分,不同浓度的HM-3在小鼠体内不同组织中的代谢分布不尽相同。在视网膜组织中,三种浓度的HM-3药物都在3h达到最大药物浓度,且药物的半衰期也无明显差异。在RPE/脉络膜复合体中,在10mg/kg组小鼠RPE/脉络膜复合体中HM-3的达峰时间为124h,而20mg/kg组小鼠RPE/脉络膜复合体中HM-3的达峰时间为124h；R_PE/脉络膜复合体中HM-3的达峰时间为116h。在血浆组织中,不同浓度给药组小鼠视网膜内的HM-3达到最大药物浓度的时间无明显差异,但低浓度的20mg/kgHM-3的代谢出现了两个药物浓度高峰,达峰时间分别是在6h和168h左右。高浓度组40mg/kgHM-3药物则呈现出单峰现象,达峰时间为124h左右。结论：HM-3能够抑制激光诱导的脉络膜新生血管的发生和发展,并可能通过与整合素αvβ3特异性结合,激活ERK1/2信号途径来调节其下游的炎症和新生血管因子的表达起作用。HM-3在小鼠体内代谢符合非房室模型,玻璃体腔注射药物可显著延长药物的代谢时间,增加药物的眼部浓度。由此我们推断HM-3可能成为治疗湿性年龄相关性黄斑病变AMD,抑制CNV的有效药物,并为药物向临床应用转化提供基础依据。
[Abstract]:Objective: age related macular degeneration (Age-related macular, degeneration, AMD) is the first cause of blindness in western developed countries, and choroidal neovascularization (choroidal neovascularization, CNV) is the leading cause of wet AMD patients with severe and irreversible visual impairment of the main reason. In recent years, China's AMD incidence increased year by year at present. The pathogenesis of AMD is still unclear, but more and more studies show that the occurrence and development of vascular endothelial growth factor and integrin signaling pathway involved in CNV, including the extracellular regulated protein kinase (extracellular regulated protein kinases, ERK) and the phosphorylation of ERK, a variety of signaling molecules, these signaling pathways play a network. In the pathological process of CNV in.HM-3 is an important drug in anti angiogenesis peptides by China Medicine University independent research and development, not only has the endothelial The active fragment of endostatin, RGD sequence and more can prime specific combination of integration, not only can inhibit angiogenesis through endothelial function, can also participate in the integrin signaling pathway, and through which to inhibit angiogenesis in vitro. The previous study has demonstrated that HM-3 has anti-inflammatory, antioxidant, anti-tumor, anti angiogenesis the new multi functions. In this study, through cooperation with the China Medicine University, the main drug for the treatment of CNV the effects and possible mechanisms of HM-3, drugs of vitreous in addition, also to give HM-3 intravitreal drug analysis in intraocular and systemic metabolic kinetics in mice, provide the basis for new targeted therapy for CNV HM-3 drug development become.
Methods: This study was divided into two parts. The first part, we use the laser of C57BL/6 mice induced by CNV model. After modeling, the treatment group after injection of 10,20,40mg/kg/HM-3 vitreous body drug, negative control group received injection of saline, the positive control group received intravitreal injection of Avastin drugs. In seventh days after modeling, the laser, the dextran perfusion fluorescence labeling, RPE- measurement of choroidal neovascularization on the area of CNV; by fluorescence angiography to evaluate CNV leakage; seventh days after modeling, the histological evaluation of laser injury in newborn blood infiltration tube; at the same time respectively by fluorescence quantitative PCR method for detection of vascular endothelial growth factor (vascularendothelialgrowthfactor, VEGF), tumor necrosis factor alpha (tumor necrosis, factor, TNF) expression in retinal pigment epithelium choroid complex; enzyme linked immunosorbent assay (E Nzyme-Linked ImmunoSorbent Assay, ELISA) expression levels of VEGF and TNF-a detection of retinal pigment epithelium choroid complex. Through Western blotting, expression of ERK1/2 and p-ERK1/2 of RPE choroid complex. In the second part, we use the HM-3 medicine glass cavity injection of different concentration, concentration was 10,20,40mg/kg, selected 12 time points were respectively 3,6,12,24,48,72,96120144168192 and 216h, the mice were sacrificed after enucleation of eyeball and blood reserve (heparin), isolated intact retina and choroid tissue RPE- layer, using indirect competitive ELISA method was used to detect the drug metabolism parameters in different time in three different tissues.
Results: in the first part, compared with saline control group, HM-3 treatment group CNV area was significantly reduced (P0.05), CNV (P0.001) also significantly reduced leakage, infiltration and 20mg/kgHM-3 drug group and positive control group Avastin were similar to.HM-3 treatment can significantly inhibit the laser injury neovascularization (P0.05) choroidal laser damage. In the expression of VEGF can be inhibited by HM-3 (P0.05), HM-3 TNF-a can inhibit RPE- in choroid (P0.05) expression and activation of ERKl/2 and p-ERK1/2 signal of laser damage early pigment epithelium choroid complex pathway (P0.05). In the second part, distribution of metabolites of different concentrations of HM-3 in different tissues mice are not the same. In the retina, drug HM-3 three concentration reaches the maximum drug concentration in 3h, and the half-life of the medication and no significant difference in the RPE/ choroid complex in 10mg/kg mice, R HM-3 in the PE/ choroid complex in the peak time was 124h, HM-3 and 20mg/kg mice in the RPE/ choroid complex in the peak time is 124h; the HM-3 R_PE/ choroid complex in the peak time of 116h. in the plasma tissue, no significant difference between the different concentrations of drug treated mice in the retina of HM-3 reached the big drug the concentration of the time, but the low concentration of 20mg/kgHM-3 metabolites appeared two concentration peak, the peak time is respectively in 6h and 168h. The high concentration group 40mg/kgHM-3 drugs showed a unimodal phenomenon, the peak time is about 124h.
Conclusion: HM-3 can inhibit the occurrence and development of choroidal neovascularization induced by laser, and may through 3 specific binding with integrin alpha v beta, expression of inflammation and angiogenesis factor activation of ERK1/2 signaling pathway to regulate its downstream effect of.HM-3 metabolism in mice with non compartmental model, intravitreal injection of drugs significantly prolong the time of drug metabolism, increase eye concentrations of drugs. We conclude that HM-3 may be the treatment of wet age-related macular degeneration AMD, effective drug to inhibit CNV, and transformed into drugs to provide the basis for clinical application.

【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R773.4

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