Rap1对实验性脉络膜新生血管的干预及作用机制研究

发布时间：2018-03-03 17:12

本文选题：脉络膜新生血管　切入点：氧化应激　出处：《河北医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:脉络膜新生血管是许多眼底疾病共有的临床表现和病理性改变,对患者的中心视力造成不可逆性损伤。脉络膜新生血管的确切发病机理尚不明确,治疗方面仍有许多问题尚待解决。我们前期研究证实,活化Rap1在实验性CNV视网膜色素上皮细胞内的表达较正常组织内减少,表明Rap1与CNV的形成有关。在此基础上,又进一步通过体内激活Rap1观察其对RPE屏障完整性以及CNV的作用,证明GTP-Rap1在CNV的生成中的重要角色。那么活化Rap1如何加强RPE屏障功能进而减少CNV形成呢?近年研究发现,GTP-Rap1可与NADPH氧化酶亚基p22phox结合进而抑制NADPH氧化酶诱导ROS的产生。此外有作者观察到,应用抗氧化剂apocynin通过抑制ROS对RPE屏障完整性的破坏进而减少CNV的形成。我们设想,活化Rap1是否通过抑制ROS对RPE屏障的破坏作用来抑制CNV形成的呢?本实验利用激光诱导建立BN大鼠CNV模型,通过向玻璃体腔注射8cpt-cAMP激活Rap1,以apocynin为抗氧化剂,观察GTP-Rap1是否通过抑制氧化应激通路来增强RPE屏障完整性从而减少CNV生成,探讨GTP-Rap1抑制实验性CNV形成的作用机制。方法:1分组:健康棕色挪威(Brown Norway,BN)大鼠60只,雌雄不限,8-10周,经眼科检查双眼底及前节均未见明显异常,随机分为3组,分别为激光模型组、玻璃体腔注射8cpt-c AMP组(8CPT组)及腹腔注射apocynin组(APO组),每组20只BN大鼠(40只眼)。2 CNV模型建立:氪激光(激光参数:波长647nm,光斑直径200μm,功率260mW,曝光时间0.05s),围绕视盘在视网膜大血管之间均匀光凝9-10个点,当看到视网膜光凝处有气泡产生可证明Bruch膜被击穿,成功建立CNV模型。3给药:激光造模后8CPT组立即玻璃体腔注射8cpt-cAMP,注射剂量为1μl。激光造模之前APO组腹腔注射apocynin,注射剂量为:0.1ml(10mg/kg/d),连续5天。观察时间:激光后5天。4real-timepcr:激光后5天,三组中随机各选取5只bn大鼠(10只眼),腹腔麻醉后立即摘除大鼠眼球,real-timepcr检测p22phox/nox4、occludinmrna水平,将上述指标进行组间比较。5westernblot:激光后5天,从三组中随机各选取5只bn大鼠(10只眼),腹腔麻醉后立即摘除大鼠眼球,westernblot检测p22phox/nox4、occludin蛋白表达水平,将上述指标进行组间比较。6荧光探针(dcfh-da)及流式细胞仪:激光后5天,从三组中随机各选取5只bn大鼠(10只眼)。腹腔麻醉后立即摘除大鼠眼球,制作rpe单细胞悬液,利用荧光探针(dcfh-da)装载rpe细胞,激光扫描共聚焦显微镜观察rpe细胞内荧光强度,流式细胞仪检测rpe细胞内ros水平,将上述指标进行组间比较。7脉络膜血管铺片:激光后5天,从三组中随机各选取5只bn大鼠(10只眼)。腹腔麻醉后颈动脉注射1ml异硫氰酸荧光素-葡聚糖,将bn大鼠眼球分离出,制作脉络膜血管铺片完成后,激光扫描共聚焦显微镜观察cnv形成情况,通过测量cnv的形成面积来进行组间比较。结果:1抗氧化剂apocynin抑制rpe-脉络膜-巩膜组织中的p22phox/nox4表达。激光后5天,p22phox/nox4mrna和蛋白表达apo组较激光模型组减少,差异有统计学意义(t=6.834,p0.001;t=6.717,p0.001)。2活化的rap1抑制rpe-脉络膜-巩膜组织中的p22phox/nox4表达。激光后5天,p22phox/nox4mrna和蛋白表达8cpt组较激光模型组减少,差异有统计学意义(t=7.834,p0.001;t=6.533,p0.001)。3抗氧化剂apocynin增加rpe-脉络膜-巩膜组织中的occludin表达。激光后5天,occludinmrna和蛋白表达apo组较激光模型组增多,差异有统计学意义(t=-5.772,p0.001;t=-6.763,p0.001)。4活化的rap1增加rpe-脉络膜-巩膜组织中的occludin表达。激光后5天,occludinmrna和蛋白表达8cpt组较激光模型组增多,差异有统计学意义(t=-5.797,p0.001;t=-6.480,p0.001)。5抗氧化剂apocynin抑制rpe细胞内的ros水平。激光后5天,激光扫描共聚焦显微镜观察,ros在激光模型组呈绿色强荧光,apo组呈绿色弱荧光。流式细胞仪检测rpe细胞内ros表达水平,与激光模型组相比APO组明显减少,差异有统计学意义(t=5.927,P0.001)。6活化的Rap1抑制RPE细胞内的ROS水平。激光后5天,激光扫描共聚焦显微镜观察,ROS在激光模型组呈绿色强荧光,8CPT组呈绿色弱荧光。流式细胞仪检测RPE细胞内ROS表达水平,与激光模型组相比8CPT组呈减少趋势,差异有统计学意义(t=6.197,P0.001)。7抗氧化剂apocynin抑制CNV生成面积。激光后5天,CNV生成面积,APO组与激光模型组相比减少,差异有统计学意义(t=8.601,P0.001)。8活化的Rap1抑制CNV生成面积。激光后5天,CNV生成面积,8CPT组与激光模型组相比呈减少趋势,差异有统计学意义(t=7.034,P0.001)。结论:GTP-Rap1可以增强RPE屏障完整性,减少实验性CNV的形成,其机制可能与抑制氧化应激产生的ROS相关。
[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.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R773.4

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