多波长激光诱导豚鼠形觉剥夺性高度近视脉络膜新生血管模型建立的研究

发布时间：2018-12-17 16:45

【摘要】：目的：探讨多波长激光诱导的形觉剥夺性高度近视豚鼠脉络膜新生血管模型建立的可行性。方法：1.多波长激光击破Bruch’s膜：3周大雄性双眼屈光参差大于6.00D三色豚鼠9只（18眼）分为激光后7d,14d,28d三组，双眼A-Scan测量眼轴，取每只鼠近视屈光度更高眼作为实验眼，另一侧眼为对照眼。采用多波长激光（532nm）击破Bruch’s膜对豚鼠双眼行视网膜光凝，激光功率、光斑直径、曝光时间分别为400mW、50μm、0.1s，每只眼在视盘上方行8个光凝斑点。于光凝后三个时间点行眼底彩照和吲哚青绿血管造影（indocyanine green angiography, ICGA），检查后处死豚鼠，摘取眼球制作标本，进行脉络膜铺片及组织病理学观察。 2.多波长激光不击破Bruch’s膜：3周大雄性双眼屈光参差小于2.00D三色豚鼠30只，在右眼面罩法形觉剥夺诱导近视6周后，多波长激光（532nm）不击破Bruch’s膜双眼光凝，分为光凝后7d,14d,28d三组，激光功率为120mW，直径200μm，曝光时间为0.1s，每只眼在视盘上方行10个光凝斑点。各组分别于光凝后三个时间点行眼底彩照和ICGA，检查后处死豚鼠，摘取眼球制作标本，进行脉络膜铺片及组织病理学观察，并通过免疫组化等分子生物学方法分析VEGF，MMP-2等因子在实验眼和对照眼视网膜等组织的蛋白表达情况。结果：1.光凝后7d出现CNV，14d达高峰，28d出现瘢痕化。以ICGA激光斑荧光充盈为准，14天时造模成功率为66.7%，且此时光镜下可见视网膜下CNV形成，脉络膜铺片可见激光斑边缘呈网簇状高荧光斑，半定量分析三个时间点CNV面积，实验眼CNV面积均大于对照眼，且14d时，实验眼CNV面积最大，28d时，，双眼CNV面积均减小。 2.面罩法形觉剥夺4周后诱导出-6.00D的近视，戴面罩眼眼轴较对照组明显增长，且主要是玻璃体腔延长。激光光凝后，ICGA三个时间点激光斑均可见；脉络膜铺片和光镜下均可见在视网膜下7d出现CNV，14dCNV达高峰，28d出现瘢痕化；免疫组化结果显示形觉剥夺眼VEGF在脉络膜毛细血管处高表达，MMP-2在视网膜色素上皮层处表达更多。结论：多波长激光击破Bruch’s膜可以诱导豚鼠CNV，并且屈光度高的豚鼠实验眼CNV面积比屈光度低的对照眼大，可能是由于屈光度高的实验眼眼轴增长导致视网膜变薄，从而为新生血管生长提供了更多的缺血缺氧等条件。多波长激光不击破Bruch’s膜可以在已经诱导出高度近视的豚鼠眼上诱导出CNV，VEGF和MMP-2参与了高度近视CNV的形成过程。这与人类病理性近视CNV发病机制较为相似，该模型可用于病理性近视CNV病因学、发病机制及药物治疗效果的研究。
[Abstract]:Objective: to investigate the feasibility of multiwavelength laser induced choroidal neovascularization model in guinea pigs with high myopia of form deprivation. Method 1: 1. Bruch's film was broken by multi-wavelength laser: 9 male anisometropia > 6.00D (18 eyes) were divided into three groups (7 days, 14 days and 28 days after laser). The axial axis of the eyes was measured by A-Scan. Each rat myopia diopter higher eye as experimental eye, the other side of the eye as a control eye. The retinal photocoagulation, laser power, spot diameter, and exposure time of the Bruch's film were observed in guinea pig eyes by using a multi-wavelength laser (532nm) method. The laser power, spot diameter and exposure time were 400mW ~ (50) 渭 m ~ (-1) ~ (0.1) s, respectively. Eight photocoagulation spots were performed in each eye over the optic disc. The guinea pigs were sacrificed at three time points after photocoagulation by fundus photography and indole green angiography (indocyanine green angiography, ICGA),). The eyeball specimens were removed and the choroidal smears and histopathology were observed. 2. The multiwavelength laser did not break the Bruch's film: the anisometropia of 3 weeks old male eyes was less than 2.00D in 30 guinea pigs. After the myopia was induced by right eye mask form deprivation for 6 weeks, the multiwavelength laser (532nm) did not break the Bruch's film binocular photocoagulation. They were divided into three groups: laser power 120 MW, diameter 200 渭 m, exposure time 0.1 s. Each eye was given 10 spots above the optic disc. Three time points after photocoagulation, the guinea pigs were killed by fundus colorimetry and ICGA, examination respectively. The eyeballs were taken out to make specimens, choroidal smears and histopathology were observed, and VEGF, was analyzed by immunohistochemistry and other molecular biological methods. Expression of MMP-2 and other factors in retinal tissues of experimental eyes and control eyes. Results: 1. CNV,14d peaked at 7 days after photocoagulation, and scarring occurred on 28 days after photocoagulation. On the basis of ICGA laser spot fluorescence filling, the successful rate of modeling was 66.7% at 14 days, and the formation of subretinal CNV was observed under light microscope, and the laser spot edge of choroidal patch was shown as a reticular high fluorescence spot. The area of CNV was semi-quantitatively analyzed at three time points. The CNV area of the experimental eyes was larger than that of the control eyes, and the CNV area of the experimental eyes was the largest at 14 days, and the CNV area of both eyes decreased at 28 days. 2. After 4 weeks of face mask deprivation, -6.00D myopia was induced. The ocular axis in mask was significantly larger than that in the control group, and the vitreous cavity was lengthened. After laser photocoagulation, laser spots were observed at three time points of ICGA, and CNV,14dCNV reached the peak at 7 days under retina and scarred at 28 days. Immunohistochemical results showed that VEGF was highly expressed in choroidal capillaries in form-deprived eyes, and MMP-2 was more expressed in retinal pigment epithelium. Conclusion: the area of CNV in guinea pig experimental eyes with high diopter is larger than that in experimental eyes with low diopter, which may be due to the retinal thinning caused by the axial growth of experimental eyes with high diopter. This provides more conditions for neovascularization, such as ischemia and hypoxia. Multiwavelength laser without breaking the Bruch's membrane can induce CNV,VEGF and MMP-2 to participate in the formation of CNV in the eyes of guinea pigs that have already induced high myopia. This model is similar to the pathogenesis of human pathological myopia (CNV). This model can be used to study the etiology, pathogenesis and therapeutic effect of CNV in pathological myopia.
【学位授予单位】：青海大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R778.11

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