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  本文關(guān)鍵詞： 脈絡(luò)膜新生血管 HM-3 整合素 信號通路 代謝動力學(xué)　出處：《南京醫(yī)科大學(xué)》2014年博士論文　論文類型：學(xué)位論文

【摘要】：目的：年齡相關(guān)性黃斑變性(Age-related macular degeneration, AMD)是西方發(fā)達(dá)國家首位的致盲眼病,而脈絡(luò)膜新生血管(choroidal neovascularization,CNV)是導(dǎo)致濕性AMD患者嚴(yán)重和不可逆的視力損傷的最主要原因。近年來,我國AMD的發(fā)病率逐年升高。目前AMD的病理機(jī)制仍不明確,但是越來越多的研究顯示血管內(nèi)皮生長因子系統(tǒng)及整合素信號通路參與CNV的發(fā)生和發(fā)展,包括細(xì)胞外調(diào)節(jié)蛋白激酶(extracellular regulated protein kinases, ERK)和磷酸化的ERK在內(nèi)的多種信號分子,這些信號途徑構(gòu)成的網(wǎng)絡(luò),在CNV的病理過程中起著重要的作用。HM-3藥物是由中國藥科大學(xué)自主研發(fā)的一種抗新生血管多肽,不僅具有內(nèi)皮抑素的活性片段,更有能與整合素特異性結(jié)合的RGD序列,不僅可以通過內(nèi)皮抑素的作用抑制新生血管,還可參與整合素信號通路,并通過其起到抑制新生血管的作用。前期的體外實(shí)驗(yàn)研究也已經(jīng)顯示HM-3具有抗炎,抗氧化,抗腫瘤,抗血管新生等多種功效。本項(xiàng)研究通過與中國藥科大學(xué)合作,主要探討HM-3藥物玻璃體腔給藥對治療CNV的作用及可能機(jī)制,此外,也對玻璃體腔給予HM-3藥物在小鼠眼內(nèi)及全身的代謝動力學(xué)進(jìn)行分析,為HM-3藥物開發(fā)成為治療CNV的新型多靶點(diǎn)藥物提供基礎(chǔ)。 方法：本項(xiàng)研究分兩部分進(jìn)行。第一部分,我們采用激光誘導(dǎo)C57BL/6小鼠產(chǎn)生CNV模型,造模后,治療組經(jīng)玻璃體注射10,20,40mg/kg/HM-3藥物,陰性對照組給予注射生理鹽水,陽性對照組給予玻璃體腔注射Avastin藥物。在激光造模后的第7天,采用熒光標(biāo)記的葡聚糖血管灌注,測量RPE-脈絡(luò)膜鋪片上CNV的面積；通過熒光血管造影來評價(jià)CNV的滲漏；造模后第7天,采用組織學(xué)來評估激光損傷部位新生血管的浸潤；同時分別通過熒光定量PCR方法檢測血管內(nèi)皮生長因子(vascularendothelialgrowthfactor, VEGF),腫瘤壞死因子-α(tumor necrosis factor, TNF)在視網(wǎng)膜色素上皮-脈絡(luò)膜復(fù)合體的表達(dá)；酶聯(lián)免疫吸附測定法(Enzyme-Linked ImmunoSorbent Assay, ELISA)檢測視網(wǎng)膜色素上皮-脈絡(luò)膜復(fù)合體中VEGF和TNF-a的表達(dá)水平。通過Western blotting,檢測視網(wǎng)膜色素上皮—脈絡(luò)膜復(fù)合體中ERK1/2及p-ERK1/2的表達(dá)。第二部分,我們采用玻璃體腔注射不同濃度的HM-3藥物,濃度分別為10,20,40mg/kg,選取12個時間點(diǎn)分別為分別為3,6,12,24,48,72,96,120,144,168,192和216h,處死小鼠后,摘除眼球并留取血液備用(肝素抗凝),分離出完整的視網(wǎng)膜和RPE-脈絡(luò)膜組織層,采用間接競爭ELISA法分別檢測在不同時間三種不同組織中的藥物代謝參數(shù)。 結(jié)果：第一部分,與生理鹽水對照組相比,HM-3治療組CNV面積顯著減少(P0.05)、CNV滲漏也顯著減少(P0.001),且20mg/kgHM-3藥物組與陽性對照Avastin組結(jié)果相似。HM-3治療能顯著抑制激光損傷部位新生血管的浸潤(P0.05)。激光損傷的脈絡(luò)膜中,VEGF的表達(dá)可以被HM-3所抑制(P0.05),此外HM-3能抑制RPE-脈絡(luò)膜中TNF-a(P0.05)的表達(dá),同時激活激光損傷早期色素上皮-脈絡(luò)膜復(fù)合體中ERKl/2和p-ERK1/2信號途徑(p0.05)。第二部分,不同濃度的HM-3在小鼠體內(nèi)不同組織中的代謝分布不盡相同。在視網(wǎng)膜組織中,三種濃度的HM-3藥物都在3h達(dá)到最大藥物濃度,且藥物的半衰期也無明顯差異。在RPE/脈絡(luò)膜復(fù)合體中,在10mg/kg組小鼠RPE/脈絡(luò)膜復(fù)合體中HM-3的達(dá)峰時間為124h,而20mg/kg組小鼠RPE/脈絡(luò)膜復(fù)合體中HM-3的達(dá)峰時間為124h；R_PE/脈絡(luò)膜復(fù)合體中HM-3的達(dá)峰時間為116h。在血漿組織中,不同濃度給藥組小鼠視網(wǎng)膜內(nèi)的HM-3達(dá)到最大藥物濃度的時間無明顯差異,但低濃度的20mg/kgHM-3的代謝出現(xiàn)了兩個藥物濃度高峰,達(dá)峰時間分別是在6h和168h左右。高濃度組40mg/kgHM-3藥物則呈現(xiàn)出單峰現(xiàn)象,達(dá)峰時間為124h左右。 結(jié)論：HM-3能夠抑制激光誘導(dǎo)的脈絡(luò)膜新生血管的發(fā)生和發(fā)展,并可能通過與整合素αvβ3特異性結(jié)合,激活ERK1/2信號途徑來調(diào)節(jié)其下游的炎癥和新生血管因子的表達(dá)起作用。HM-3在小鼠體內(nèi)代謝符合非房室模型,玻璃體腔注射藥物可顯著延長藥物的代謝時間,增加藥物的眼部濃度。由此我們推斷HM-3可能成為治療濕性年齡相關(guān)性黃斑病變AMD,抑制CNV的有效藥物,并為藥物向臨床應(yīng)用轉(zhuǎn)化提供基礎(chǔ)依據(jù)。
[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.

【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R773.4

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本文編號：1537398