【作者】 张翠英；

【导师】 牟国营；

【作者基本信息】 山东大学 ， 临床医学（眼科学）（专业学位）， 2018， 博士

【摘要】 第一部分兔真菌性角膜炎动物模型制作研究背景:真菌是一大类真核细胞型微生物。细胞核高度分化,有核膜和核仁,胞浆内有完整的细胞器。自然界存在的真菌种类很多,目前发现对人有致病性真菌和机会致病性真菌已超过百种。真菌性角膜炎是一种感染性角膜疾病,致盲率非常高,是角膜盲的重要原因。角膜的各种外伤为真菌的感染提供了机会,抗生素、皮质激素及抗病毒药物的使用增多,角膜接触镜的广泛使用,致使眼部真菌病的发病率呈上升趋势。真菌主要分为丝状菌和酵母菌(主要为念珠菌属)两大类;目前的真菌性角膜炎研究中,主要和白色念珠菌、镰刀菌属、曲霉菌属相关,少见暗色真菌。角膜真菌感染多见于农民或户外工作人群,其中角膜外伤是最主要的原因,其他包括长期使用激素或者抗生素,过敏性结膜炎,配戴接触镜,全身免疫力低下等。观察真菌性角膜炎的特点和研究真菌性角膜炎治疗的有效性,首先需要成功建立合适的动物模型。兔眼形态及解剖与人眼相近且角膜较大,便于手术操作和观察;此外,兔性情温和,容易取得,易于喂养,故本实验选择兔作为实验动物。目前国际上通用的真菌性角膜炎的动物模型是向兔角膜基质内注射真菌孢子致深层角膜真菌感染的模型。向兔角膜深层基质内注射真菌孢子的方法,感染成功率高,发生角膜混合感染的几率底,成本低廉且操作方便,但缺点是容易出现角膜穿孔引起眼内感染,并且角膜刮片检查难以取到深部感染的组织。在本实验动物模型制作中采用角膜浅基质层注射真菌菌液的方法,感染成功率高并且明显减少了角膜穿孔的风险,且角膜刮片容易取得感染的组织,但必须在眼科手术显微镜下轻柔操作并注意充分麻醉。目的:建立兔浅层真菌性角膜炎动物模型,探讨模型制作过程中的注意事项,观察感染前后角膜及其他眼部变化,并通过角膜刮片培养及共聚焦显微镜检查明确真菌感染及感染菌种。方法:健康成年新西兰大白兔48只,体重2.5～3.0kg,雌雄均用,排除眼前段活动性疾病,预饲养两周。实验兔随机分为三组,分别为:360组,440组和空白对照组。48只实验兔均采用右眼为模型眼,A型超声角膜测厚仪测定兔眼角膜中央厚度。用10%水合氯醛腹腔注射麻醉(3ml/kg),右眼用盐酸奥布卡因滴眼液(倍诺喜)表面麻醉3次,固定头部;在眼科手术显微镜下,开睑器开睑后,生理盐水冲洗结膜囊,并将提前制备好的浓度为106CFU/ml(Colony-Forming Units,CFU,单位体积中的活菌个数)标准白色念珠菌菌液0.1ml注入角膜中央部浅层基质内,形成直径4mm圆形灰白色区域。制作兔角膜白色念珠菌感染的模型后,通过裂隙灯检查、角膜溃疡刮片培养鉴定菌种、共聚焦显微镜观察真菌。结果:A型超声角膜测厚仪测定兔眼角膜中央厚度,各组间角膜中央厚度无显著性差异。裂隙灯检查角膜真菌感染:白色念珠菌感染后1天,角膜溃疡形成,角膜轻度水肿,能透见虹膜文理,前房无积浓;感染后3天,角膜溃疡灶面积增大,周边可见卫星灶,角膜水肿弥漫性加重,不能透见虹膜;感染后5天,溃疡灶面积进一步扩大,溃疡灶和卫星灶融合,并有苔垢样坏死组织附着,溃疡边缘稍隆起毛糙不齐,角膜弥漫性雾状水肿加重,前房可见积脓。角膜刮片培养:菌落为圆形,较大,表面中间粗糙起皱,假菌丝浸入培养基内,为类酵母菌型菌落典型表现。革兰染色阳性,着色不均匀,可见菌细胞呈圆形或卵圆形,并可见假菌丝。无染色高倍镜可见真菌细胞出芽生成假菌丝,假菌丝不分枝,长短不一;使用YST卡进行菌株鉴定菌种鉴定为白色念珠菌。共聚焦显微镜检查:模型制作后第3～5天,角膜溃疡形成,多次共聚焦显微镜检查,所有模型兔均查见菌丝或孢子,共聚焦显微镜检查可见大量真菌菌丝和炎细胞,菌丝细长弯曲成团索样,高折光。结论:1.兔眼与人眼形态及解剖相近,眼球较大,是感染性角膜炎理想的动物选择。2.在本实验动物模型制作中采用角膜浅基质层注射真菌的方法,成功制作了兔角膜白色念珠菌性角膜炎动物模型,其操作简单、成功率高、角膜刮片容易,并且明显减少了角膜穿孔的风险。第二部分360nmUVA或440nm蓝光联合核黄素角膜胶原交联对兔真菌性角膜炎的疗效观察研究背景:真菌性角膜炎是一种致盲率高的严重角膜感染性疾病,近40年来发病率明显增加。目前主要的治疗方式仍然是抗真菌药物局部或联合全身应用,由于大多数抗真菌药物眼组织穿透力小且毒性较大,药物治疗相对棘手。另外角膜溃疡刮除联合碘烧灼也有应用,但效果有限。胶原的高机械强度与胶原结构有密切的关系。角膜胶原交联疗法的基本原理是根据光敏剂核黄素的独特的吸收光谱,在紫外线或可见光的照射下,吸收光子能量,诱导胶原纤维和内部化学基团之间,形成更多的共价键交联,从而增加了胶原生物力学特性,提高了胶原纤维的机械强度和抵抗角膜扩张的能力,增加胶原对酶降解的抵抗力。紫外线A(Ultraviolet Radiation A,UVA)设备使用发光二极管产生的紫外线A波长在360～380nm,此设备包含聚焦系统、焦点距离、光线直径和束均匀度等参数,操作者能根据情况调节参数的大小。角膜胶原交联在眼科应用广泛,特别是对于圆锥角膜和角膜扩张等疗效肯定。核黄素是一种具有3个环状结构的极性分子,具有吸附核酸的特点,能够插入DNA或者RNA碱基内。0.1%核黄素能产生最大的吸收作用,90%紫外线辐射能在角膜基质中被吸收,最大限度保护了角膜内皮,晶状体和视网膜。核黄素在角膜胶原交联过程中具有双重作用,它不但是诱导交联的光敏剂;作为选择性滤波器,核黄素还能够保护下面的组织免受UVA的损伤。另外,足量的核黄素使体内酸性物质减少,不利于真菌生长,因此患者抵御真菌感染的能力增强。360nmUVA联合核黄素角膜交联治疗(corneal cross-lingking,CXL)对真菌性角膜炎的疗效,在动物实验及临床上都得到了证实。我们知道核黄素的吸收波峰包括360nm和440nm,目前核黄素联合360nm UVACXL治疗对真菌性角膜炎的治疗作用已经得到确认,但是核黄素联合440nm蓝光CXL治疗对真菌性角膜炎的疗效尚无人观察。目的:本实验采用兔真菌性角膜炎为动物模型实验的方法,观察分析360nmUVA或440nm蓝光联合核黄素CXL治疗对兔真菌性角膜炎的疗效。方法:兔真菌性角膜炎动物模型48只,已随机分为空白对照组,360组,440组。空白对照组不做任何处理,360组和440组分别行角膜CXL治疗。实验兔用10%水合氯醛腹腔注射麻醉(3ml/kg),右眼用盐酸奥布卡因滴眼液(倍诺喜)表面麻醉3次,固定头部,开睑器开睑,用圆钝刀片刮除直径9.0mm的中央角膜上皮层和表层溃疡组织。0.1%核黄素溶液冲洗角膜,2分钟1次,连续半小时;裂隙灯可观察到前房黄染,确定核黄素充分渗透角膜进入前房。360组采用0.1%核黄素点眼30分钟后,360nmUVA照射30分钟;440组采用0.1%核黄素点眼30分钟后,440nm蓝光照射30分钟;总能量设定均为3mW/cm2,照射距离均为5cm。治疗后1、3、7、15天分别对各组兔进行裂隙灯观察、角膜照相并行溃疡面积计算分析、共聚焦显微镜检查比较。CXL治疗后1、7,15天天各组分别耳缘动脉注气处死4只实验兔,立即剪取其角膜并甲醛固定,切片染色后进行病理观察分析。结果:治疗前各组间溃疡面积比较无统计学意义,治疗后1天各组间比较无统计学意义(P>0.05);治疗后3天,空白对照组较治疗前增大6.94%,360组角膜溃疡面积减小35.65%,P=0.012;440组角膜溃疡面积减小37.94,P=0.015,数据分析均有显著性差异;360组和440组间比较P=0.88,数据分析没有显著性差异。360组和440组分别与空白对照组比较p<0.05,数据分析具有显著性差异。治疗后7天,空白对照组较治疗前减小13.14%,360组角膜溃疡面积减小73.82%,P=0.00;440组角膜溃疡面积减小70.00%,P=0.000,数据分析均有显著性差异;360组和440组间比较P=0.75,数据分析没有显著性差异,360组和440组分别与空白对照组比较p<0.01,数据分析具有显著性差异。治疗后15天,空白对照组溃疡面积减小83.16%;360组核和440组角膜溃疡消失。共聚焦显微镜检查:治疗后1天各组间无明显差异;治疗后3,7天360组和440组较空白对照组真菌菌丝减少,真菌菌丝变短棒样,继而消失,角膜中的炎症细胞较空白对照组减少。治疗后15天,空白对照组可见角膜基质细胞增大增多,炎症细胞和少量真菌菌丝呈短棒状高反光;360组和440组未见菌丝及孢子,可见增大的基质细胞。病理检查结果:CXL治疗后1天:各组均可见角膜炎症反应重,局部角膜上皮缺失,可见溃疡形成,局部可见脓肿灶,周围较多淋巴细胞、中性粒细胞浸润及嗜酸性粒细胞。治疗后7天:空白对照组:角膜坏死严重,溃疡形成,局部角膜穿孔,炎症反应重;360组角膜炎症反应相对较轻,角膜水肿,散在淋巴细胞、嗜酸性粒细胞及少量中性粒细胞浸润,纤维细胞母细胞及小血管增生,未见明显坏死及溃疡形成;440组角膜炎症反应相对较轻,角膜水肿,散在淋巴细胞、嗜酸性粒细胞及少量中性粒细胞浸润,纤维细胞母细胞及小血管增生,未见明显坏死及溃疡形成。治疗后15天:空白对照组角膜溃疡不明显,局部角膜上皮缺失,角膜轻水肿,散在淋巴细胞、嗜酸性粒细胞及少量中性粒细胞浸润,纤维细胞母细胞及小血管增生。360和440组未见角膜溃疡,角膜上皮完整,可见纤维母细胞增生。结论:360nmUVA和440nm蓝光联合核黄素CXL对兔真菌性角膜炎的治疗均有效,治疗效果无明显差异。更多还原

【Abstract】 Part IBuilding rabbit model of fungal keratitisBackground:Fungal keratitis is an infective corneal disease with very high blind rate,and is an important cause of corneal blindness.The industrial injuries of the cornea provided opportunities for fungal infection;in addition,with the wide application of antibiotics,corticosteroids,and anti-viral drugs,as well as the increased application of corneal contact lens,the incidence of fungal keratitis increases by years.Fungi mainly include filamentous fungi(such as fusarium,aspergillus,penicillium,and dematiaceous fungi)and yeasts(mainly candida species).Candida albicans is the major pathogenic fungi in the developed countries and cold regions.The previously published studies about fungal keratitis also mainly focused on candida albicans,fusarium,aspergillus,and very few dematiaceous fungi.Inducing appropriate animal model of fungal keratitis is essential to investigate the effectiveness of the treatments for fungal keratitis.Rabbit is a very commonly used experimental animal for ophthalmic researches,and has been acknowledged as a better living animal model than other animals.The morphologyand anatomic features of the rabbit eyes are similar to human eyes,the eyeballs are relatively big,and the area of the cornea is about 30%of the eyeball area,which could facilitate the surgical processes and the observations.Moreover,rabbit is a tame animal that is easy to raise,and is very cost-effective,therefore the present study selected rabbit to induce the fungal keratitis model.Currently,the universally used method to induce fungal keratitis model is to inject fungal spores into the corneal stroma of the rabbits to induce the deep corneal fungal infection.Injecting fungal spores into the corneal stroma is with high successful rate of infection,while the rate of mixed corneal infection is very low,which is also very cheap and easy to perform.Objectives:To induce a rabbit model of superficial fungal keratitis,identify the key processes in the model induction,observe the corneal changes before and after the infection,and clarify the fungal infection and the type of fungi infected via corneal scraping followed by culture and confocal microscopy.Methods:Forty-eight healthy adult New Zealand White Rabbits with the body weight of 2.5-3.0 kg and either sex were obtained.After rabbits with active eye diseases of anterior segment were excluded,the rabbits were acclimated for 2 weeks.106 CFU/mL standard candida albicans solution was prepared,and then 0.1 mL of the solution was injected into the superficial corneal stroma to form a round gray-white change with the diameter of about 4 mm.and thus induce the rabbit model of candida albicans infection.Slit-lamp examination,corneal ulcer scraping followed by culture to clarify the infected fungus,and confocal microscope to observe the fungi were performed.Results:Slit-lamp examination of the corneal fungal infection showed the formation of corneal ulcer and corneal edema,with satellite lesions around.Corneal ulcer scraping followed by culture showed the fungal clone was round and relatively big,the center of the clone surface was rough and wrinkled,and the pseudohypha extended into the culture medium,which is the typical feature of yeast-like fungi clone.Confocal microscope observation revealed that corneal ulcer appeared at 3-5 days after the model induction.Repeated confocal microscope observations showed that all the rabbits were with hyphae or spores.Conclusion:I)The morphology and anatomic features of the rabbit eyes are similar to human eyes,the eyeballs are relatively large,and thus is the ideal animal for the induction of infective keratitis model;2)In the present study,fungus was injected into the superficial comeal stroma to induce the rabbit model,which successfully induced the rabbit model of candida keratitis.This method is simple,while the successful rate is very high,and the risk of corneal perforation is significantly reduced.Part ⅡObservation of curative effect regarding corneal cross-linking treatment of riboflavin combined with 360nmUVA or 440 nm blue-light for fungal keratitis of the rabbitsBackground:Fungal keratitis is a serious eye disease with high blind rate.Fungal keratitis is now the first leading infective keratitis in some regions in China,and the incidence of this disease increased evidently in the past 40 years.Currently,the major treatment method for fungal keratitis is still the local or combined with systemic application of antifungal drugs.However,as most of the antifungal drugs are with relatively high toxicity and low ocular tissue permeability,thus drug treatment for fungal keratitis is relatively difficult.In addition,scraping of the corneal ulcer in combination with iodine burning is also used,but the effectiveness is limited.The basic rational of corneal cross-linking is the unique absorption spectrum of riboflavin.the photosensitizer,which could absorb the photon energy under ultraviolet or visible light irradiation,induce the chemical groups in and between the collagens to form more covalent bond crosslinks,thus increase the biomechanical characteristics of the collagens,and improve the mechanical strength of the collagens and the ability to against the dilation of the cornea.Corneal collagen cross-linking has been widely used in the clinical practices in the Department of Ophthalmology,especially for the treatment of keratoconus and corneal dilation,of which the treatment efficacies have already been demonstrated.The efficacies of using 360 nm UVA combining riboflavin to induce corneal collagen cross-linking in treating fungal keratitis have already been demonstrated in both animal experiments and the clinical practices.It is well known that the absorption spectrum of riboflavin peaks at 360 and 440 nm.Currently,the effectiveness of inducing corneal cross-linking(CXL)with riboflavin combining 360 nm ultraviolet radiation A(UVA)has already been acknowledged,while the effectiveness of inducing CXL with riboflavin combining 440 nm UVA has not been investigated to date.Objective:To investigate the effectiveness of using 360 nm UVA or 440 nm blue light in combination with riboflavin to induce corneal collagen cross-linking in treating fungal keratitis in rabbit models.Methods:Forty-eight rabbit models of corneal keratitis were randomly divided into control,360 and 440 groups.The rabbits in the control group did not receive any treatment.while the ones in the 360 and 440 groups were treated with corneal crosslinking.For the rabbits in the 360 group.the rabbits were dripped with 0.1%riboflavin for 30 min.and then irradiated with 360 nm UVA for 30 min:while for the ones in the 440 group.the rabbits were dripped with 0.1%riboflavin for 30 min.and then irradiated with 440 nm blue licght for 30 min.Slit-lamp examinations.photographing of the cornea,and confocal microscopy were performed on the days 1,3.7and15 after the treatment,and the areas of the corneal ulcers were calculated.On the days 1,7,15 after the treatment,4 rabitts were executed respectively every group,then sniped the cornea and made pathological section.Results:No significant difference was found among the three groups on the day 1 after the treatment(P>0.05).On the day 3 after the treatment,the ulcer area in the control group increased by 6.94%,while the area in the 360 and 440 groups decreased by 35.65%(P=0.012)and 37.94%(P=0.015)as compared with the area before the treatment,respectively.On the day 7 after the treatment,the ulcer area in the control group decreased by 13.14%,while the area in the 360 and 440 groups decreased by 73.82%(P=0.00)and 70.00%(P=0.001)as compared with the area before the treatment,respectively.There is significant difference between the 360 and control group,between the 440 and control group.However,no significant difference between the 360 nm and 440 nm groups on the day 7 after the treatment was found(P=0.75).On the day 15 after the treatment,the ulcer area in the control group decreased by 83.16%,360 and 440 group corneal ulcers are recured.Confocal microscopy results showed no evident difference among the three groups on the day 1 after the treatment;while on the days 3 and 7 after the treatment,the numbers of the fungal hyphae in the 360 nm and 440 nm groups were evidently lower than that in the control group,the fungal hyphae changed to short-bar like morphology and then disappeared,the numbers of inflammatory cells were also lower than that in the control group.On the day 15 after treatment,there are a little numbers fungal hyphaes and corneal stroma cells;And there are not fungal hyphae in the 360 and 440 group.Pathological section report:On the day 1 after treatment,inflammatory reaction was serious in every group.On the day 7 after treatment,infammatory reaction in the control group was more serious,but it’s better in the 360 nm and 440 nm groups.On the day 15 after treatment.there are more fibroblasts and small vessels in all groups.But epithelium corneae is not complete in the control group.Conclusion:Using both 360 nm UVA and 440 nm blue light in combination with riboflavin to induce corneal collagen cross-linking are effective in treating rabbit fungal keratitis,which showed no significant difference between these two methods.更多还原