【作者】 王庆华；

【导师】 施勤；

【作者基本信息】 苏州大学 ， 免疫学， 2014， 硕士

【摘要】 目的：通过研究阿瓦斯汀(Avastin)结膜下给药对兔眼碱烧伤角膜新生血管的影响,探索阿瓦斯汀治疗角膜新生血管的安全性及其有效性,为临床应用阿瓦斯汀治疗角膜新生血管性疾病提供实验依据。方法：选用成年新西兰雄性大白兔60只(苏州大学动物实验中心),体重在2-3Kg之间,健康状况良好,均取左眼作为实验眼,随机抽选45只兔左眼用1mol/LNaOH溶液烧伤,建立碱烧伤角膜新生血管模型。未造模的15只兔左眼设为A正常对照组,成功造模的45只兔再随机分为三组,每组15只,分别为B模型对照组(造模成功后不做治疗),C新药干预组(术后2小时结膜下注射2.5mg/0.1ml阿瓦斯汀),D常规治疗照组(术后2小时结膜下注射地塞米松0.5mg/0.1ml),于碱烧伤后4、7、14天拍照记录,观察活体兔眼结膜充血、角膜水肿浑浊、上皮的完整性及新生血管的生长情况,定量计算角膜新生血管面积,并于三个时间点每组随机取5只兔用空气栓塞法处死,取实验眼角膜行病理检查及免疫组化检测角膜VEGF和CD31的表达。结果：1、碱烧伤4天,造模组所有兔眼均出现明显的炎症反应,结膜充血水肿,角膜水肿浑浊,上皮不完整,B组角膜缘见少许新生血管长入,C、D组未发现有角膜新生血管,7天,造模组所有兔眼结膜充血明显,角膜新生血管生长旺盛,自角膜缘呈毛刷样向烧伤区爬行,10-14点区域新生血管出现最多。14天,角、结膜充血水肿减轻,角膜上皮修复,角膜浑浊,中央烧伤区最为致密,角膜新生血管有所消退。2、病理显示,正常对照组角膜上皮完整,层次清晰,角膜基质纤维排列整齐,无炎细胞及血管,术后4天造模组角膜上皮连续性中断,上皮下及浅基质层见少量新生血管芽,管腔少量血细胞,大量炎细胞主要聚集于角膜浅层,7天时角膜新生血管密集,并向深基质层长入,管腔粗大且充满血细胞。14天时,炎细胞减少,表面新生血管部分消退,基质层见管径粗大的血管,血细胞充盈,在不同时间点,C、D组之间差别无统计学差异,C、D组与B组之间比较有统计学差异。3、角膜VEGF检测结果：碱烧伤4天,造模组所有兔眼角膜均出现VEGF,7天VEGF表现强阳性,14天时密度降低,三个时间点统计,C、D组之间、/EGF表达强度无统计学差异,C、D组VEGF表达强度较B组轻,与B组之间比较有统计学差异。4、角膜CD31检测结果示：CD31在正常兔眼角膜组织中无表达,造模组兔角膜中均有表达,7天时表达强阳性,各时间点C、D治疗组阳性细胞数明显低于模型对照组,差异有统计学意义。结论：1、兔眼角膜碱烧伤4天角膜开始有新生血管长入,烧伤后7天时结膜炎症反应比较重,角膜水肿浑浊,新生血管生长旺盛,14天结膜炎症反应减轻,角膜新生血管有所消退。2、阿瓦斯汀与地塞米松对兔眼碱烧伤后形成的新生血管都有很好的抑制作用,与B造模组相比,C、D组在三个不同时间点检测到的新生血管、VEGF及CD31均显著降低,C组与D组在三个不同的时间点检测的各项指标无显著差异。3、阿瓦斯汀治疗兔眼角膜新生血管简单、安全、效果显著,在观察期内未见并发症发生。更多还原

【Abstract】 Objective The purpose of our study is, by subconjunctival administration ofAvastin in the rabbit with corneal neovascularization caused by ocular alkali burns, toexplore the safety and effectiveness of Avastin in the treatment of cornealneovascularisation, and further to provide experimental evidence of clinical applicationof Avastinto treat ocular diseases with corneal neovascularisation.Method60male New Zealand white rabbits were purchased and selectedfromexperimental animal farm of Soochow University. All of the rabbits were weighingbetween2-3kgwith good health. Only left eyes were included.45left eyes wererandomly selected toestablish alkali burn corneal neovascularization model by using1mol/LNaOH solution. The remaining15left eyes were set as normal control group(Group A). The45left eyeswith successfully induced corneal neovascularisation wererandomly divided into three groups (n=15), namely Group B (model control group,successful model without treatment), Group C (new drug intervention group,subconjunctival injection of Avastinwith2.5mg/0.1ml two hours after modeling), andGroup D (conventional treatment group, subconjunctival injection of dexamethasonewith0.5mg/0.1ml two hours after modeling). Camera recording was taken at4,7, and14days after alkali burn. Conjunctival hyperemia, corneal edema, opacity, integrity andgrowth of new blood vessels in the corneal epithelium were observed in vivo, and thearea of corneal neovascularisation were calculated quantitatively. Five rabbits wererandomly selected to be killed by air embolism at the three time points in eachexperimental group, whose left corneas were taken to make pathological examinationand to detect VEGF and CD31expression by Immunohistochemistry.Results1. After four days’ alkali burns, all of the experimental eyes revealedconjunctivalhyperemia and edema; corneal edema, opacity and epithelial damage. In the group B,a small amount of tiny new blood vessels grown into the cornea at limbus. However, inthe group C and D, there was no obvious new blood vessel. At7days, all of theexperimental eyes revealedheavier conjunctival hyperemia, limbal vascular networkcongestion. In addition, brush-like new blood vessels grown vigorously from thelimbuscrawling up to the burned area, with amounts of new blood vessels at10to14o’clock. At7days,conjunctival congestion alleviated, corneal edema absorbed to someextent, epithelial repaired, the whole cornea opacified with more dense in the centralcornea, and corneal neovascularization subsided. Compared with group B, conditions ofconjunctival hyperemia, corneal edema and neovascularization were significantlyreduced in the group C and D at these three time points.2. Pathological examination showed intact corneal epithelial cells, clear layer, andcorneal stromal fibers arranging in neat rows without vascular tissue in the normalcorneas. After four days’ alkali burns, corneal thickening, epithelial discontinuity, andlarge number of inflammatory cells infiltration under the corneal epithelia and in thesuperficial corneal stroma (mainly gathered in the superficial cornea) were found. Asmall part of new blood vessels were found at some regions of limbus. At7days, thenew blood vessels were dense at limbus and grown into the deep corneal stroma to burnarea with wide lumen and being full of blood cells. At14days, the amounts ofinflammatory cells reduced, new blood vessels subsided to some extent, but still withwide lumen and being full of blood cells. At different time points, there was nosignificant difference between Group C and D, but significant differences were foundbetween Group C/D and B.3. Corneal VEGF test results: After four days’ alkali burns, VEGFwere detectedinall of the experimental corneas. At7days, the VEGF expression was strongly positive.But at14days,the density of VEGF decreased. At different time points, there was nosignificant difference between Group C and D, but significant differences were foundbetween Group C/D and B.4. Corneal CD31test results: No expression of CD31was detected in normal rabbitcornea tissue, but all experimental corneas expressed, with strong positive expression at7days. At each time point, positive cells in the treatment group (Group C and D) wassignificantly lower than the model control group (Group B). The differences werestatistically significant. Conclusion1. Both Avastin and dexamethasone have good inhibition effect on ocularneovascularization caused by alkali burns in rabbits. Compared with model controlgroup (Group B), the conditions of neovascularization, VEGF and CD31weresignificantly lower in the Group C and D at three different time points. However, herewas no significant difference between Group C and D.2. Avastinis a simple, safe, and effective drug to treat cornealneovascularization inthe rabbit, without significant complications during the observation period.更多还原