本文選題：脈絡(luò)膜新生血管　切入點(diǎn)：氧化應(yīng)激　出處：《河北醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:脈絡(luò)膜新生血管是許多眼底疾病共有的臨床表現(xiàn)和病理性改變,對(duì)患者的中心視力造成不可逆性損傷。脈絡(luò)膜新生血管的確切發(fā)病機(jī)理尚不明確,治療方面仍有許多問題尚待解決。我們前期研究證實(shí),活化Rap1在實(shí)驗(yàn)性CNV視網(wǎng)膜色素上皮細(xì)胞內(nèi)的表達(dá)較正常組織內(nèi)減少,表明Rap1與CNV的形成有關(guān)。在此基礎(chǔ)上,又進(jìn)一步通過體內(nèi)激活Rap1觀察其對(duì)RPE屏障完整性以及CNV的作用,證明GTP-Rap1在CNV的生成中的重要角色。那么活化Rap1如何加強(qiáng)RPE屏障功能進(jìn)而減少CNV形成呢?近年研究發(fā)現(xiàn),GTP-Rap1可與NADPH氧化酶亞基p22phox結(jié)合進(jìn)而抑制NADPH氧化酶誘導(dǎo)ROS的產(chǎn)生。此外有作者觀察到,應(yīng)用抗氧化劑apocynin通過抑制ROS對(duì)RPE屏障完整性的破壞進(jìn)而減少CNV的形成。我們?cè)O(shè)想,活化Rap1是否通過抑制ROS對(duì)RPE屏障的破壞作用來抑制CNV形成的呢?本實(shí)驗(yàn)利用激光誘導(dǎo)建立BN大鼠CNV模型,通過向玻璃體腔注射8cpt-cAMP激活Rap1,以apocynin為抗氧化劑,觀察GTP-Rap1是否通過抑制氧化應(yīng)激通路來增強(qiáng)RPE屏障完整性從而減少CNV生成,探討GTP-Rap1抑制實(shí)驗(yàn)性CNV形成的作用機(jī)制。方法:1分組:健康棕色挪威(Brown Norway,BN)大鼠60只,雌雄不限,8-10周,經(jīng)眼科檢查雙眼底及前節(jié)均未見明顯異常,隨機(jī)分為3組,分別為激光模型組、玻璃體腔注射8cpt-c AMP組(8CPT組)及腹腔注射apocynin組(APO組),每組20只BN大鼠(40只眼)。2 CNV模型建立:氪激光(激光參數(shù):波長(zhǎng)647nm,光斑直徑200μm,功率260mW,曝光時(shí)間0.05s),圍繞視盤在視網(wǎng)膜大血管之間均勻光凝9-10個(gè)點(diǎn),當(dāng)看到視網(wǎng)膜光凝處有氣泡產(chǎn)生可證明Bruch膜被擊穿,成功建立CNV模型。3給藥:激光造模后8CPT組立即玻璃體腔注射8cpt-cAMP,注射劑量為1μl。激光造模之前APO組腹腔注射apocynin,注射劑量為:0.1ml(10mg/kg/d),連續(xù)5天。觀察時(shí)間:激光后5天。4real-timepcr:激光后5天,三組中隨機(jī)各選取5只bn大鼠(10只眼),腹腔麻醉后立即摘除大鼠眼球,real-timepcr檢測(cè)p22phox/nox4、occludinmrna水平,將上述指標(biāo)進(jìn)行組間比較。5westernblot:激光后5天,從三組中隨機(jī)各選取5只bn大鼠(10只眼),腹腔麻醉后立即摘除大鼠眼球,westernblot檢測(cè)p22phox/nox4、occludin蛋白表達(dá)水平,將上述指標(biāo)進(jìn)行組間比較。6熒光探針(dcfh-da)及流式細(xì)胞儀:激光后5天,從三組中隨機(jī)各選取5只bn大鼠(10只眼)。腹腔麻醉后立即摘除大鼠眼球,制作rpe單細(xì)胞懸液,利用熒光探針(dcfh-da)裝載rpe細(xì)胞,激光掃描共聚焦顯微鏡觀察rpe細(xì)胞內(nèi)熒光強(qiáng)度,流式細(xì)胞儀檢測(cè)rpe細(xì)胞內(nèi)ros水平,將上述指標(biāo)進(jìn)行組間比較。7脈絡(luò)膜血管鋪片:激光后5天,從三組中隨機(jī)各選取5只bn大鼠(10只眼)。腹腔麻醉后頸動(dòng)脈注射1ml異硫氰酸熒光素-葡聚糖,將bn大鼠眼球分離出,制作脈絡(luò)膜血管鋪片完成后,激光掃描共聚焦顯微鏡觀察cnv形成情況,通過測(cè)量cnv的形成面積來進(jìn)行組間比較。結(jié)果:1抗氧化劑apocynin抑制rpe-脈絡(luò)膜-鞏膜組織中的p22phox/nox4表達(dá)。激光后5天,p22phox/nox4mrna和蛋白表達(dá)apo組較激光模型組減少,差異有統(tǒng)計(jì)學(xué)意義(t=6.834,p0.001;t=6.717,p0.001)。2活化的rap1抑制rpe-脈絡(luò)膜-鞏膜組織中的p22phox/nox4表達(dá)。激光后5天,p22phox/nox4mrna和蛋白表達(dá)8cpt組較激光模型組減少,差異有統(tǒng)計(jì)學(xué)意義(t=7.834,p0.001;t=6.533,p0.001)。3抗氧化劑apocynin增加rpe-脈絡(luò)膜-鞏膜組織中的occludin表達(dá)。激光后5天,occludinmrna和蛋白表達(dá)apo組較激光模型組增多,差異有統(tǒng)計(jì)學(xué)意義(t=-5.772,p0.001;t=-6.763,p0.001)。4活化的rap1增加rpe-脈絡(luò)膜-鞏膜組織中的occludin表達(dá)。激光后5天,occludinmrna和蛋白表達(dá)8cpt組較激光模型組增多,差異有統(tǒng)計(jì)學(xué)意義(t=-5.797,p0.001;t=-6.480,p0.001)。5抗氧化劑apocynin抑制rpe細(xì)胞內(nèi)的ros水平。激光后5天,激光掃描共聚焦顯微鏡觀察,ros在激光模型組呈綠色強(qiáng)熒光,apo組呈綠色弱熒光。流式細(xì)胞儀檢測(cè)rpe細(xì)胞內(nèi)ros表達(dá)水平,與激光模型組相比APO組明顯減少,差異有統(tǒng)計(jì)學(xué)意義(t=5.927,P0.001)。6活化的Rap1抑制RPE細(xì)胞內(nèi)的ROS水平。激光后5天,激光掃描共聚焦顯微鏡觀察,ROS在激光模型組呈綠色強(qiáng)熒光,8CPT組呈綠色弱熒光。流式細(xì)胞儀檢測(cè)RPE細(xì)胞內(nèi)ROS表達(dá)水平,與激光模型組相比8CPT組呈減少趨勢(shì),差異有統(tǒng)計(jì)學(xué)意義(t=6.197,P0.001)。7抗氧化劑apocynin抑制CNV生成面積。激光后5天,CNV生成面積,APO組與激光模型組相比減少,差異有統(tǒng)計(jì)學(xué)意義(t=8.601,P0.001)。8活化的Rap1抑制CNV生成面積。激光后5天,CNV生成面積,8CPT組與激光模型組相比呈減少趨勢(shì),差異有統(tǒng)計(jì)學(xué)意義(t=7.034,P0.001)。結(jié)論:GTP-Rap1可以增強(qiáng)RPE屏障完整性,減少實(shí)驗(yàn)性CNV的形成,其機(jī)制可能與抑制氧化應(yīng)激產(chǎn)生的ROS相關(guān)。
[Abstract]:Objective: choroidal neovascularization is many fundus diseases common clinical manifestations and pathological changes, causing irreversible damage to the central visual acuity of patients. The exact pathogenesis of choroidal neovascularization is not clear, for there are still many problems to be solved. Our previous studies demonstrated that activation of Rap1 expression in epithelial cells of experiment CNV in the retinal pigment than normal tissue decreased, indicating the formation of Rap1 and CNV. On this basis, further by in vivo activation of Rap1 was observed on the RPE barrier integrity and the role of CNV and GTP-Rap1 in the formation of CNV proved an important role in the activation of Rap1 RPE. So how to strengthen the barrier function and reduce CNV form? Recent research found that GTP-Rap1 can inhibit NADPH oxidase induced by ROS combined with NADPH oxidase subunit p22phox. In addition the author observed that the application of anti oxidation Agent apocynin on RPE barrier integrity damage and reduce the formation of CNV by inhibiting ROS activation. We assume that whether Rap1 can inhibit the destruction of ROS RPE barrier to suppress CNV formation? This experiment using laser induced to establish BN rat model of CNV, by intravitreal injection of 8cpt-cAMP activated Rap1, apocynin antioxidants, to observe whether GTP-Rap1 by inhibiting the oxidative stress pathway to enhance RPE barrier integrity and reduce CNV generation, to explore the mechanism of GTP-Rap1 inhibition of CNV formation. Methods: 1 groups: healthy brown Norway (Brown Norway, BN) 60 rats, male or female, 8-10 weeks after ophthalmic examination and bottom eyes the previous section showed no obvious abnormalities, were randomly divided into 3 groups, respectively, laser model group, intravitreal injection of 8cpt-c AMP group (group 8CPT) and intraperitoneal injection of apocynin group (APO group), 20 rats in each group of BN rats (40 eyes) of.2 CNV. A: krypton laser (laser: wavelength 647nm, spot diameter of 200 m, power 260mW, exposure time 0.05s), around the optic disc between the retinal vessels of uniform photocoagulation 9-10 points when retinal photocoagulation with bubbles that Bruch film breakdown, successfully established the CNV model.3 administration the laser group immediately after modeling 8CPT intravitreal injection of 8cpt-cAMP, the dose was 1 before L. laser model group APO apocynin intraperitoneal injection, injection quantity: 0.1ml (10mg/kg/d), for 5 consecutive days. The observation time: 5 days after 5 days after laser.4real-timepcr: laser, the three groups randomly selected 5 bn the rats (10 eyes), intraperitoneal anesthesia immediately after the rat with the eye, real-timepcr detection of p22phox/nox4, occludinmrna level, the indexes were compared between the two groups.5westernblot: 5 days after laser, from the three groups randomly selected 5 BN rats (10 eyes), were picked immediately after anesthesia In addition to the rat eyeball, Westernblot detection of p22phox/nox4, occludin protein expression, the above indexes were compared between the two groups of.6 fluorescent probe (DCFH-DA) and flow cytometry: 5 days after laser, from the three groups randomly selected 5 BN rats (10 eyes). After intraperitoneal anesthesia immediately removed the rat eyeball RPE, single cell suspension, using fluorescent probe (DCFH-DA) loaded RPE cells, fluorescence intensity was observed in RPE cells with laser scanning confocal microscope, to detect the level of ROS in RPE cells by flow cytometry, the above indexes were compared between the two groups of.7 choroidal vascular preparation: 5 days after laser, from the three groups randomly selected 5 BN rats (10 eyes) were anesthetized. Carotid artery injection of 1ml fluorescein isothiocyanate dextran, BN rats were isolated from the eye, making the choroidal vascular preparations completed, microscope into CNV shaped confocal laser scanning, by measuring the CNV shape Into the area were compared between the two groups. Results: 1 apocynin inhibited the antioxidant rpe- choroid sclera tissue. The expression of p22phox/nox4 in 5 days after laser, p22phox/nox4mrna and protein expression in apo group compared with the laser model group decreased, the difference was statistically significant (t=6.834, p0.001; t=6.717, p0.001).2 activated Rap1 inhibited rpe- choroid the scleral tissue of p22phox/nox4 expression. 5 days after laser, p22phox/nox4mrna and protein expression in 8cpt group compared with the laser model group decreased, the difference was statistically significant (t=7.834, p0.001; t=6.533, p0.001).3 apocynin rpe- to increase the antioxidant of sclera and choroid. The expression of occludin in 5 days after laser, occludinmrna and protein expression in apo group than in the laser model group increased, the difference was statistically significant (t=-5.772, p0.001; t=-6.763, p0.001).4 activated Rap1 increased rpe- of sclera choroid in occludin expression. 5 days after laser, occludinmrna And the protein expression of 8cpt group compared with the laser model group increased, the difference was statistically significant (t=-5.797, p0.001; t=-6.480, p0.001).5 antioxidant apocynin inhibited RPE intracellular ROS level. 5 days after laser, laser scanning confocal microscopy, ROS showed green laser Qiang Yingguang in the model group, apo group showed a weak green fluorescence flow. Cytometry was used to detect the expression of ROS in RPE cells, compared with the model group, APO laser group were significantly reduced, the difference was statistically significant (t=5.927, P0.001).6 activated Rap1 inhibited RPE cells. The level of ROS in 5 days after laser, laser scanning confocal microscopy, ROS showed strong green fluorescence in the laser model group. The 8CPT group showed green fluorescence. Flow cytometry was used to detect the expression level of ROS in RPE cells compared with model group, laser group 8CPT decreased, the difference was statistically significant (t=6.197, P0.001).7 apocynin antioxidants inhibit the formation of CNV area. 5 days after laser, CNV APO laser generating area, group and model group decreased, the difference was statistically significant (t=8.601, P0.001).8 activated Rap1 inhibited the production of CNV area. 5 days after laser, CNV laser and generation area, 8CPT group compared to the model group decreased, the difference was statistically significant (t=7.034. P0.001). Conclusion: GTP-Rap1 can enhance the RPE barrier integrity, reduce the formation of experimental CNV, its mechanism may be related to the inhibition of oxidative stress produced by ROS.

【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】：2017
【分類號(hào)】：R773.4

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