【摘要】：目的:脈絡(luò)膜新生血管(choroidal noevascularization, CNV)與眼部許多疾病有關(guān),是引起視力障礙的重要原因之一。近期研究發(fā)現(xiàn),眼部的炎癥反應(yīng)和免疫活化在CNV的發(fā)生和發(fā)展中起著重要作用。 塞來(lái)昔布是一種常見的非甾體類消炎藥,具有COX-2特異抑制的作用。炎癥刺激可誘導(dǎo)COX-2生成,從而導(dǎo)致炎性PG類物質(zhì)的合成和聚積,尤其是前列腺素,引起炎癥反應(yīng)。塞來(lái)昔布可通過抑制COX-2阻止PG類物質(zhì)的產(chǎn)生,達(dá)到抗炎、鎮(zhèn)痛及退熱作用。 本文采用激光建立CNV動(dòng)物模型,通過觀察CNV中TLR4、NF-κB、COX-2和VEGF的變化規(guī)律,以及COX-2抑制劑塞來(lái)昔布對(duì)CNV的抑制作用,從而了解CNV中炎癥對(duì)新生血管的影響,進(jìn)一步探討CNV的發(fā)病機(jī)制和治療方法。 方法: 1實(shí)驗(yàn)性脈絡(luò)膜新生血管中TLR4、NF-κB表達(dá)規(guī)律的研究 1.1 10只健康棕色挪威大鼠,隨機(jī)分為空白對(duì)照組、激光組,激光組以波長(zhǎng)647 nm的氪激光(波長(zhǎng)647 nm,光斑直徑100μm ,功率120 mW,曝光時(shí)間0.05s),以視盤為中心圍繞視盤均勻光凝9個(gè)點(diǎn),以見到有氣泡產(chǎn)生提示Bruch膜被擊穿為準(zhǔn)建立大鼠CNV模型。 1.2激光組大鼠于光凝后7d進(jìn)行眼底熒光造影(FFA),觀察CNV形成情況。 1.3兩組大鼠分別于光凝后3、7、14、21、30d隨機(jī)選取一只,處死動(dòng)物,摘除眼球取眼后段組織制作石蠟切片。常規(guī)HE染色及光鏡觀察CNV的形成情況。并做TLR4、NF-κB免疫組化染色,采用HPIAS-1000型高清晰度彩色病理圖文分析系統(tǒng)測(cè)定其染色的平均灰度值,觀察這兩個(gè)因子的表達(dá)變化情況。 2塞來(lái)昔布對(duì)實(shí)驗(yàn)性脈絡(luò)膜新生血管中TRL4、NF-κB、COX-2和VEGF表達(dá)影響及其意義的研究 2.1 BN大鼠15只,隨機(jī)分為空白對(duì)照組、實(shí)驗(yàn)對(duì)照組、塞來(lái)昔布治療組。治療組通過灌胃法給藥(塞來(lái)昔布50 mg/kg,一天兩次),1周后實(shí)驗(yàn)對(duì)照組和治療組均給予激光光凝(方法同1)建立大鼠CNV模型。各組大鼠分別于光凝后第3、7、14、21、30 d進(jìn)行熒光素眼底血管造影。 2.2造影后6h待大鼠體內(nèi)熒光素染料大部分被排出,處死動(dòng)物,摘除一只眼球(另一只眼球用做RT-PCR反應(yīng)的標(biāo)本)制作空白對(duì)照組球后段組織切片及實(shí)驗(yàn)對(duì)照組和治療組的“最大CNV膜”切片。常規(guī)HE染色及用免疫組化法分別檢測(cè)各組TRL4、NF-κB、COX-2和VEGF在脈絡(luò)膜中的表達(dá)。采用HPIAS-1000型高清晰度彩色病理圖文分析系統(tǒng)測(cè)定其染色的平均灰度值。 2.3用上述留取的各組大鼠其中一只眼球進(jìn)行逆轉(zhuǎn)錄聚合酶鏈反應(yīng),來(lái)檢測(cè)上述三組中脈絡(luò)膜組織TRL4、NF-κB、COX-2和VEGF mRNA變化。 結(jié)果: 1實(shí)驗(yàn)性脈絡(luò)膜新生血管中TLR4、NF-κB表達(dá)規(guī)律的研究 1.1光凝后7 d,FFA光凝斑早期輕度熒光素滲漏,晚期滲漏明顯,證實(shí)CNV形成。 1.2光鏡下可見,Bruch膜破裂,在光凝區(qū)周圍有巨噬細(xì)胞浸潤(rùn),RPE細(xì)胞遷移和纖維母細(xì)胞增生等,在視網(wǎng)膜下可見完整的CNV管腔。 1.3免疫組化結(jié)果顯示,空白對(duì)照組TLR4、NF-κB在脈絡(luò)膜中的表達(dá)比較弱。在激光組中,TLR4、NF-κB隨時(shí)間的發(fā)展,表達(dá)呈先上升后下降的趨勢(shì)。它們均在光凝后3 d明顯增加,7 d時(shí)達(dá)高峰。 2塞來(lái)昔布對(duì)實(shí)驗(yàn)性脈絡(luò)膜新生血管中TRL4、NF-κB、COX-2和VEGF表達(dá)影響及其意義的研究 2.1實(shí)驗(yàn)對(duì)照組和治療組中,兩組FFA進(jìn)行比較,CNV均在第21d時(shí)達(dá)高峰,但治療組CNV發(fā)生率明顯低于實(shí)驗(yàn)對(duì)照組(P0.01)。 2.2免疫組化結(jié)果顯示,在實(shí)驗(yàn)性CNV中,COX-2、VEGF和TRL4、NF-κB都有明顯表達(dá),都隨時(shí)間的發(fā)展,表達(dá)先呈上升而后下降的趨勢(shì)。TRL4、NF-κB在光凝后3 d明顯增加,7 d時(shí)達(dá)高峰;COX-2、VEGF在光凝后7 d明顯表達(dá)增多,21 d時(shí)達(dá)高峰。光凝后CNV表達(dá)高峰期7 d和21d時(shí),TLR4、NF-κB和COX-2、VEGF在治療組中比在實(shí)驗(yàn)對(duì)照組中的表達(dá)明顯降低,差異具有統(tǒng)計(jì)學(xué)意義(P0.01)。2.3 RT-PCR顯示TLR4、NF-κB、COX-2、VEGF mRNA表達(dá)。在試驗(yàn)對(duì)照組和治療組中,四個(gè)基因在轉(zhuǎn)錄水平上的表達(dá)變化同免疫組化結(jié)果類似。 結(jié)論: 1實(shí)驗(yàn)性CNV的發(fā)病機(jī)制有可能是通過TLR4介導(dǎo)產(chǎn)生免疫反應(yīng),從而使NF-κB的信號(hào)通路活化,誘導(dǎo)血管生長(zhǎng)因子的表達(dá)。 2預(yù)防性服用選擇性COX-2抑制劑塞來(lái)昔布能夠有效抑制激光誘導(dǎo)CNV的生成,推測(cè)其機(jī)制可能是通過抑制NF-κB表達(dá)進(jìn)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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R773.4

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