塞来昔布对实验性脉络膜新生血管TLR4、NF-κB、COX-2和VEGF表达影响的研究

发布时间：2018-08-24 15:42

【摘要】：目的:脉络膜新生血管(choroidal noevascularization, CNV)与眼部许多疾病有关,是引起视力障碍的重要原因之一。近期研究发现,眼部的炎症反应和免疫活化在CNV的发生和发展中起着重要作用。塞来昔布是一种常见的非甾体类消炎药,具有COX-2特异抑制的作用。炎症刺激可诱导COX-2生成,从而导致炎性PG类物质的合成和聚积,尤其是前列腺素,引起炎症反应。塞来昔布可通过抑制COX-2阻止PG类物质的产生,达到抗炎、镇痛及退热作用。本文采用激光建立CNV动物模型,通过观察CNV中TLR4、NF-κB、COX-2和VEGF的变化规律,以及COX-2抑制剂塞来昔布对CNV的抑制作用,从而了解CNV中炎症对新生血管的影响,进一步探讨CNV的发病机制和治疗方法。方法: 1实验性脉络膜新生血管中TLR4、NF-κB表达规律的研究 1.1 10只健康棕色挪威大鼠,随机分为空白对照组、激光组,激光组以波长647 nm的氪激光(波长647 nm,光斑直径100μm ,功率120 mW,曝光时间0.05s),以视盘为中心围绕视盘均匀光凝9个点,以见到有气泡产生提示Bruch膜被击穿为准建立大鼠CNV模型。 1.2激光组大鼠于光凝后7d进行眼底荧光造影(FFA),观察CNV形成情况。 1.3两组大鼠分别于光凝后3、7、14、21、30d随机选取一只,处死动物,摘除眼球取眼后段组织制作石蜡切片。常规HE染色及光镜观察CNV的形成情况。并做TLR4、NF-κB免疫组化染色,采用HPIAS-1000型高清晰度彩色病理图文分析系统测定其染色的平均灰度值,观察这两个因子的表达变化情况。 2塞来昔布对实验性脉络膜新生血管中TRL4、NF-κB、COX-2和VEGF表达影响及其意义的研究 2.1 BN大鼠15只,随机分为空白对照组、实验对照组、塞来昔布治疗组。治疗组通过灌胃法给药(塞来昔布50 mg/kg,一天两次),1周后实验对照组和治疗组均给予激光光凝(方法同1)建立大鼠CNV模型。各组大鼠分别于光凝后第3、7、14、21、30 d进行荧光素眼底血管造影。 2.2造影后6h待大鼠体内荧光素染料大部分被排出,处死动物,摘除一只眼球(另一只眼球用做RT-PCR反应的标本)制作空白对照组球后段组织切片及实验对照组和治疗组的“最大CNV膜”切片。常规HE染色及用免疫组化法分别检测各组TRL4、NF-κB、COX-2和VEGF在脉络膜中的表达。采用HPIAS-1000型高清晰度彩色病理图文分析系统测定其染色的平均灰度值。 2.3用上述留取的各组大鼠其中一只眼球进行逆转录聚合酶链反应,来检测上述三组中脉络膜组织TRL4、NF-κB、COX-2和VEGF mRNA变化。结果: 1实验性脉络膜新生血管中TLR4、NF-κB表达规律的研究 1.1光凝后7 d,FFA光凝斑早期轻度荧光素渗漏,晚期渗漏明显,证实CNV形成。 1.2光镜下可见,Bruch膜破裂,在光凝区周围有巨噬细胞浸润,RPE细胞迁移和纤维母细胞增生等,在视网膜下可见完整的CNV管腔。 1.3免疫组化结果显示,空白对照组TLR4、NF-κB在脉络膜中的表达比较弱。在激光组中,TLR4、NF-κB随时间的发展,表达呈先上升后下降的趋势。它们均在光凝后3 d明显增加,7 d时达高峰。 2塞来昔布对实验性脉络膜新生血管中TRL4、NF-κB、COX-2和VEGF表达影响及其意义的研究 2.1实验对照组和治疗组中,两组FFA进行比较,CNV均在第21d时达高峰,但治疗组CNV发生率明显低于实验对照组(P0.01)。 2.2免疫组化结果显示,在实验性CNV中,COX-2、VEGF和TRL4、NF-κB都有明显表达,都随时间的发展,表达先呈上升而后下降的趋势。TRL4、NF-κB在光凝后3 d明显增加,7 d时达高峰;COX-2、VEGF在光凝后7 d明显表达增多,21 d时达高峰。光凝后CNV表达高峰期7 d和21d时,TLR4、NF-κB和COX-2、VEGF在治疗组中比在实验对照组中的表达明显降低,差异具有统计学意义(P0.01)。2.3 RT-PCR显示TLR4、NF-κB、COX-2、VEGF mRNA表达。在试验对照组和治疗组中,四个基因在转录水平上的表达变化同免疫组化结果类似。结论: 1实验性CNV的发病机制有可能是通过TLR4介导产生免疫反应,从而使NF-κB的信号通路活化,诱导血管生长因子的表达。 2预防性服用选择性COX-2抑制剂塞来昔布能够有效抑制激光诱导CNV的生成,推测其机制可能是通过抑制NF-κB表达进而抑制上游因子TLR4和下游因子COX-2、VEGF。
[Abstract]:Objective: Choroidal noevascularization (CNV) is associated with many ocular diseases and is one of the important causes of visual impairment. Recent studies have shown that ocular inflammation and immune activation play an important role in the occurrence and development of CNV.
Celecoxib is a common non-steroidal anti-inflammatory drug with COX-2 specific inhibitory effect. Inflammatory stimulation can induce the production of COX-2, which leads to the synthesis and accumulation of inflammatory PG substances, especially prostaglandins, causing inflammation. Celecoxib can inhibit the production of PG substances by inhibiting COX-2 to achieve anti-inflammatory, analgesic and antipyretic effects. Use.
In order to understand the effect of inflammation on neovascularization in CNV and to further explore the pathogenesis and treatment of CNV, the animal model of CNV was established by laser irradiation.
Method:
1 expression pattern of TLR4, NF- kappa B in experimental choroidal neovascularization
1.110 healthy brown Norwegian rats were randomly divided into blank control group and laser group. The CNV model of rats was established by krypton laser with wavelength 647 nm, spot diameter 100 micron, power 120 mW, exposure time 0.05s, and uniform photocoagulation around the optic disc at 9 points.
In the 1.2 laser group, the fundus fluorescein angiography (FFA) was performed on 7d after photocoagulation, and the formation of CNV was observed.
1.3 Two groups of rats were randomly selected at 3,7,14,21 and 30 days after photocoagulation and killed. The posterior segment of the eyeball was removed and paraffin sections were made. The formation of CNV was observed by routine HE staining and light microscopy. The expression of these two factors was observed.
Effect of celecoxib on the expression of TRL4, NF-kappa B, COX-2 and VEGF in experimental choroidal neovascularization and its significance
2.1 BN rats were randomly divided into blank control group, experimental control group and celecoxib treatment group. The treatment group was given celecoxib 50 mg/kg twice a day by intragastric administration. One week later, the experimental control group and the treatment group were given laser photocoagulation (the same method 1) to establish CNV model. The rats in each group were fluoresced on the 3rd, 7th, 14th, 21st and 30th days after photocoagulation. Fundus angiography.
2.2 After 6 hours of radiography, most of the fluorescein dyes were excreted and the animals were sacrificed. One eyeball (another eyeball was used for RT-PCR reaction) was excised and the posterior segment of the eyeball in the blank control group and the "maximum CNV membrane" sections of the experimental control group and the treatment group were made. The expression of kappa B, COX-2 and VEGF in choroid was detected by HPIAS-1000 high-definition color pathological image analysis system.
2.3 The changes of TRL4, NF-kappa B, COX-2 and VEGF mRNA in choroidal tissues were detected by reverse transcription polymerase chain reaction (RT-PCR) in one of the eyeballs of the rats.
Result:
1 expression pattern of TLR4, NF- kappa B in experimental choroidal neovascularization
1.1 after photocoagulation 7 d, FFA photocoagulation spot early slight fluorescein leakage, late leakage obvious, confirmed CNV formation.
1.2 Under light microscope, Bruch membrane ruptured, macrophage infiltration, RPE cell migration and fibroblasts proliferation were found around the photocoagulation area, and intact CNV lumen could be seen under the retina.
1.3 Immunohistochemical results showed that the expression of TLR4 and NF-kappa B in the choroid of the blank control group was weaker. In the laser group, the expression of TLR4 and NF-kappa B increased first and then decreased with the development of time. They all increased significantly 3 days after photocoagulation and reached the peak 7 days after photocoagulation.
Effect of celecoxib on the expression of TRL4, NF-kappa B, COX-2 and VEGF in experimental choroidal neovascularization and its significance
2.1 In the experimental control group and the treatment group, FFA of the two groups were compared, CNV peaked at the 21st day, but the incidence of CNV in the treatment group was significantly lower than that in the experimental control group (P 0.01).
2.2 Immunohistochemical staining showed that COX-2, VEGF, TRL4 and NF-kappa B were significantly expressed in experimental CNV. The expression of COX-2, VEGF, TRL4 and NF-kappa B increased first and then decreased with the development of time. The expression of TLR4, NF-kappa B, COX-2 and VEGF in the treatment group was significantly lower than that in the experimental control group at day 21 (P 0.01).
Conclusion:
1. The pathogenesis of experimental CNV may be mediated by TLR4 to produce immune response, thus activating the signal pathway of NF-kappa B and inducing the expression of vascular growth factor.
Preventive use of selective COX-2 Inhibitor Celecoxib can effectively inhibit the production of laser-induced CNV. It is speculated that the mechanism may be through inhibiting the expression of NF-kappa B and then inhibiting the upstream factors TLR4 and downstream factors COX-2, VEGF.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R773.4

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