MIP-2在棘阿米巴性角膜炎中的表达
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摘要
目的:建立有效的棘阿米巴角膜炎动物模型,检测棘阿米巴角膜炎中巨噬细胞炎症蛋白-2(MIP-2)的表达并探讨MIP-2在棘阿米巴角膜炎发病机制中的作用,观察干扰MIP-2对棘阿米巴角膜炎的影响。
方法:1、健康加卡利亚仓鼠80只随机分为4组,每组20只。刮除仓鼠右眼角膜中央区50%的上皮,对照组覆盖无菌角膜接触镜并滴入PBS液,接触镜组覆盖有阿米巴黏附的接触镜并滴入PBS液,悬液组覆盖无菌角膜接触镜并滴入阿米巴悬液,混合组覆盖有阿米巴黏附的接触镜并滴入阿米巴悬液,在模型建立48、72小时后进行研究观察。2、健康加卡利亚仓鼠80只随机分为4组,每组20只。空白对照组不予任何处理;条件对照组刮除仓鼠右眼角膜中央区50%的上皮,覆盖无菌角膜接触镜并滴入PBS液;实验一、二组刮除右眼上皮后覆盖有阿米巴黏附的接触镜并滴入阿米巴悬液。分别于接种后1d、2d、3d、5d、7d用半定量逆转录聚合酶链反应方法(RT-PCR)检测各组中MIP-2的表达(空白对照组、条件对照组和实验一组取右眼角膜,实验二组取左眼角膜),并对不同时间点角膜组织行HE染色及光镜观察。3、健康加卡利亚仓鼠80只随机分为4组,每组20只。刮除所有仓鼠右眼角膜中央区50%的上皮,覆盖有阿米巴黏附的接触镜并滴入阿米巴悬液。空白对照组不予任何处理;条件对照组在模型制成48小时角膜内注射PBS液;实验一组注射TNF-α;实验二组注射rMIP-2。分别于接种后3d、5d、7d、12d、18d用半定量逆转录聚合酶链反应方法检测各组中MIP-2的表达,并对不同时间点角膜组织行HE染色及光镜观察。
结果:1、混合组方法能成功地建立稳定的加卡利亚仓鼠棘阿米巴角膜炎动物模型,并经角膜刮片用10%氢氧化钾封片镜检、角膜组织原虫培养和病理切片染色检查证实。2、空白对照组、条件对照组和实验二组各个观测点中均未检测到MIP-2的表达,HE染色也未发现有炎症细胞浸润。实验一组在接种阿米巴1天后即出现MIP-2的阳性表达,随时间推移表达量逐渐升高,至5天时表达最强,随后表达水平下降,但至接种后7d仍呈阳性表达。同时,光镜下观察到5个观测点均有中性粒细胞和淋巴细胞浸润,以2、3、5d为著。3、两实验组与空白对照组相比MIP-2的相对表达量有统计学意义(P＜0.05),光镜下3、5d两个观测点可以见到密度高于空白对照组的中性粒细胞浸润。两实验组全部观测点的总愈合率分别为55%和45%,高于空白对照组的15%;两个后期观察点12、18d的总愈合率分别为100%和87.5%,也高于空白对照组的37.5%。
结论:1、在去上皮的加卡利亚仓鼠角膜上覆盖与棘阿米巴原虫共同培养24小时后的减张角膜接触镜,并滴加一定量阿米巴悬液的方法,可以成功建立棘阿米巴角膜炎的动物模型,建模成功率高、病程稳定。模型制做后3天打开睑裂所得模型,虽在统计学上与2天后打开睑裂得到的模型无显著差异,但成功率略高。此方法可重复性高且模型符合该病自然病程,模型可应用于棘阿米巴角膜炎的发病机理或药物实验等研究中。2、成功的棘阿米巴角膜炎模型组第一天即发现中性粒细胞和淋巴细胞浸润,其后炎症细胞随时间推移逐渐增多,但在第七天明显下降,这一演变过程与MIP-2表达量的演变过程相符合。这表明MIP-2作为趋化因子参与了棘阿米巴角膜炎的免疫防御行为,发挥了其在自然免疫中针对中性粒细胞的特异性趋化作用。3、TNF-α可以在棘阿米巴角膜炎的发病过程中诱导MIP-2基因表达上调,高于正常表达量的MIP-2可以特异性趋化高于正常水平的中性粒细胞参与炎症反应,并能够在不严重加剧角膜破坏的情况下,加速棘阿米巴角膜炎的愈合过程。
Purpose:To establish an effective animal model of acanthamoeba keratitis in Djungarian hamster,detect the expression of macrophage inflammatory protein 2 (MIP-2) in Acanthamoeba Keratitis,investigate the function of MIP-2 in the pathogenesy of acanthamoeba keratitis and survey the changes of acanthamoeba keratitis by interfering the expression of MIP-2.
Methods:1.Eighty healthy Djungarian Hamsters used in this experiment were divided into four groups,twenty in each group.Scraped 50%epithelium of central cornea of both eyes.Control group was covered by aseptic contact lens and dropped in PBS,CL group was covered by ameba-laden contact lenses and dropped in PBS, Suspension group was covered by aseptic contact lens and dropped in suspension of Acanthamoeba,Combinated group was covered by ameba-laden contact lenses and dropped in suspension of Acanthamoeba.Investigated the clinal and pathological characteristics of acanthamoeba keratitis 48h and 72h after infection.
2、Eighty healthy Djungarian Hamsters used in this experiment were divided into four groups,twenty in each group.The blank control group was granted with no disposal. In conditional control group,Scraped 50%epithelium of central cornea of the right eye and covered by aseptic contact lens and dropped in PBS.In experimental group one and two,Scraped 50%epithelium of central cornea of the right eye and covered by ameba-laden contact lenses and dropped in suspension of Acanthamoeba.Detected the expression of MIP-2 with semi-quantitative reverse transcription polymerase chain reaction(RT-PCR) respectively at 1d、2d、3d、5d、7d after innoculation(The blank control group、the conditional control group and the experimental group one used the cornea of right eye,the experimental group two used the cornea of left eye). Took tissue of cornea at different observing points and stained them with hematoxylin and eosin(H&E).Then,Observation was made under microscope.
3.Eighty healthy Djungarian Hamsters used in this experiment were divided into four groups,twenty in each group.Scraped 50%epithelium of central cornea of the right eye and covered by ameba-laden contact lenses and dropped in suspension of Acanthamoeba.The blank control group was granted with no disposal.In conditional control group,intracorneal administration of PBS was done 48 hours after infection. Intracorneal administration of TNF-αwere done in experimental group one and intracorneal administration of rMIP-2 were done in experimental group two at the same time.Detected the expression of MIP-2 with semi-quantitative reverse transcription polymerase chain reaction(RT-PCR) respectively at3d、5d、7d、12d、18d after innoculation.Took tissue of cornea at different observing points and stained them with hematoxylin and eosin(H&E).Then,Observation was made under microscope.
Results:1.Combinated group can establish stable model of acanthamoeba keratitis successfully in Djungarian hamster.This result can be confirmed by 10%potassium hydroxide(KOH) wet mount,corneal cytology,isolation and cultivation of the amoeba,corneal biopsy and histopathological examination.2、In the blank control group、the conditional control group and the experimental group two,no expression of MIP-2 were detected at any observing points.Observation made under light microscope showed no inflammatory cell infiltration too.In the experimental group one,the positive expression of MIP-2 could be detected one day after infection.The level of MIP-2 increased with the time-lapse.The level of MIP-2 reached its peak five days after infection,soon after;its level began to decrease.But,the expression of MIP-2 remained positive seven days after infection.Meanwhile,observation made under light microscope showed there are neutrophil and lymphocyte infiltration at each observing point,especially at 2、3、5d after infection.3.The expression of MIP-2 showed the significant difference between those two experimental groups and the blank control group(P＜0.05).Observation made under light microscope showed that the inflammatory cell infiltration density of those two experimental groups was higher than that of the blank control group 3 and 5 days after infection.The total healing rates of all the observing points in those two experimental groups were 55% and 45%respectively,which were higher than 15%of the blank control group.In the later stage observing points,that is,12d and 18d after infection,the total healing rate in those two experimental groups were 100%and 87.5%respectively,which were higher than 37.5%of the blank control group.
Conclusions:1.By covering ameba-laden contact lenses on the cornea of Djungarian Hamster with preexisting lesions and dropping 20μl suspension of Acanthamoeba (3×10~6 organisms/ml) under it,can establish a successful animal mode of acanthamoeba keratitis.Modeling achievement ratio of this method is high and has a stable course of disease.Although the differences of the affectivity of animal model have no statistical significance between opening the eyelids 48h and 72h after infection,the achievement ratio of opening the eyelids 72h after infection is higher. This method has high achievement ratio ang repeatability.This model can analogue the spontaneous process of human who has infected acanthamoeba keratitis andwould facilitate investigations exploring the pathophysiology,cell biology,genetics, immunology,and therapy of this disease.
2.In effective animal model of acanthamoeba keratitis,neutrophil and lymphocyte infiltration could be detected one day after infection.Subsequently,The quantity of inflammatory cell increased with the time-lapse but began to decrease at seven days after infection.Its developing process was coincided with that of the expression of MIP-2.This result indicated that,as a chemotactic factor,MIP-2 was involved in the immune defence behaviors of acanthamoeba keratitis and played a specific chemotaxis to neutrophil in innate immunity.
3.TNF-αcould induce the up-regulation of MIP-2 gene in the invasion course of acanthamoeba keratitis.Higher expressed MIP-2 could attract more neutrophils involved in the inflammatory reaction,which could accelerate the agglutination of acanthamoeba keratitis without intensifing corneal destruction.
方法:1、健康加卡利亚仓鼠80只随机分为4组,每组20只。刮除仓鼠右眼角膜中央区50%的上皮,对照组覆盖无菌角膜接触镜并滴入PBS液,接触镜组覆盖有阿米巴黏附的接触镜并滴入PBS液,悬液组覆盖无菌角膜接触镜并滴入阿米巴悬液,混合组覆盖有阿米巴黏附的接触镜并滴入阿米巴悬液,在模型建立48、72小时后进行研究观察。2、健康加卡利亚仓鼠80只随机分为4组,每组20只。空白对照组不予任何处理;条件对照组刮除仓鼠右眼角膜中央区50%的上皮,覆盖无菌角膜接触镜并滴入PBS液;实验一、二组刮除右眼上皮后覆盖有阿米巴黏附的接触镜并滴入阿米巴悬液。分别于接种后1d、2d、3d、5d、7d用半定量逆转录聚合酶链反应方法(RT-PCR)检测各组中MIP-2的表达(空白对照组、条件对照组和实验一组取右眼角膜,实验二组取左眼角膜),并对不同时间点角膜组织行HE染色及光镜观察。3、健康加卡利亚仓鼠80只随机分为4组,每组20只。刮除所有仓鼠右眼角膜中央区50%的上皮,覆盖有阿米巴黏附的接触镜并滴入阿米巴悬液。空白对照组不予任何处理;条件对照组在模型制成48小时角膜内注射PBS液;实验一组注射TNF-α;实验二组注射rMIP-2。分别于接种后3d、5d、7d、12d、18d用半定量逆转录聚合酶链反应方法检测各组中MIP-2的表达,并对不同时间点角膜组织行HE染色及光镜观察。
结果:1、混合组方法能成功地建立稳定的加卡利亚仓鼠棘阿米巴角膜炎动物模型,并经角膜刮片用10%氢氧化钾封片镜检、角膜组织原虫培养和病理切片染色检查证实。2、空白对照组、条件对照组和实验二组各个观测点中均未检测到MIP-2的表达,HE染色也未发现有炎症细胞浸润。实验一组在接种阿米巴1天后即出现MIP-2的阳性表达,随时间推移表达量逐渐升高,至5天时表达最强,随后表达水平下降,但至接种后7d仍呈阳性表达。同时,光镜下观察到5个观测点均有中性粒细胞和淋巴细胞浸润,以2、3、5d为著。3、两实验组与空白对照组相比MIP-2的相对表达量有统计学意义(P＜0.05),光镜下3、5d两个观测点可以见到密度高于空白对照组的中性粒细胞浸润。两实验组全部观测点的总愈合率分别为55%和45%,高于空白对照组的15%;两个后期观察点12、18d的总愈合率分别为100%和87.5%,也高于空白对照组的37.5%。
结论:1、在去上皮的加卡利亚仓鼠角膜上覆盖与棘阿米巴原虫共同培养24小时后的减张角膜接触镜,并滴加一定量阿米巴悬液的方法,可以成功建立棘阿米巴角膜炎的动物模型,建模成功率高、病程稳定。模型制做后3天打开睑裂所得模型,虽在统计学上与2天后打开睑裂得到的模型无显著差异,但成功率略高。此方法可重复性高且模型符合该病自然病程,模型可应用于棘阿米巴角膜炎的发病机理或药物实验等研究中。2、成功的棘阿米巴角膜炎模型组第一天即发现中性粒细胞和淋巴细胞浸润,其后炎症细胞随时间推移逐渐增多,但在第七天明显下降,这一演变过程与MIP-2表达量的演变过程相符合。这表明MIP-2作为趋化因子参与了棘阿米巴角膜炎的免疫防御行为,发挥了其在自然免疫中针对中性粒细胞的特异性趋化作用。3、TNF-α可以在棘阿米巴角膜炎的发病过程中诱导MIP-2基因表达上调,高于正常表达量的MIP-2可以特异性趋化高于正常水平的中性粒细胞参与炎症反应,并能够在不严重加剧角膜破坏的情况下,加速棘阿米巴角膜炎的愈合过程。
Purpose:To establish an effective animal model of acanthamoeba keratitis in Djungarian hamster,detect the expression of macrophage inflammatory protein 2 (MIP-2) in Acanthamoeba Keratitis,investigate the function of MIP-2 in the pathogenesy of acanthamoeba keratitis and survey the changes of acanthamoeba keratitis by interfering the expression of MIP-2.
Methods:1.Eighty healthy Djungarian Hamsters used in this experiment were divided into four groups,twenty in each group.Scraped 50%epithelium of central cornea of both eyes.Control group was covered by aseptic contact lens and dropped in PBS,CL group was covered by ameba-laden contact lenses and dropped in PBS, Suspension group was covered by aseptic contact lens and dropped in suspension of Acanthamoeba,Combinated group was covered by ameba-laden contact lenses and dropped in suspension of Acanthamoeba.Investigated the clinal and pathological characteristics of acanthamoeba keratitis 48h and 72h after infection.
2、Eighty healthy Djungarian Hamsters used in this experiment were divided into four groups,twenty in each group.The blank control group was granted with no disposal. In conditional control group,Scraped 50%epithelium of central cornea of the right eye and covered by aseptic contact lens and dropped in PBS.In experimental group one and two,Scraped 50%epithelium of central cornea of the right eye and covered by ameba-laden contact lenses and dropped in suspension of Acanthamoeba.Detected the expression of MIP-2 with semi-quantitative reverse transcription polymerase chain reaction(RT-PCR) respectively at 1d、2d、3d、5d、7d after innoculation(The blank control group、the conditional control group and the experimental group one used the cornea of right eye,the experimental group two used the cornea of left eye). Took tissue of cornea at different observing points and stained them with hematoxylin and eosin(H&E).Then,Observation was made under microscope.
3.Eighty healthy Djungarian Hamsters used in this experiment were divided into four groups,twenty in each group.Scraped 50%epithelium of central cornea of the right eye and covered by ameba-laden contact lenses and dropped in suspension of Acanthamoeba.The blank control group was granted with no disposal.In conditional control group,intracorneal administration of PBS was done 48 hours after infection. Intracorneal administration of TNF-αwere done in experimental group one and intracorneal administration of rMIP-2 were done in experimental group two at the same time.Detected the expression of MIP-2 with semi-quantitative reverse transcription polymerase chain reaction(RT-PCR) respectively at3d、5d、7d、12d、18d after innoculation.Took tissue of cornea at different observing points and stained them with hematoxylin and eosin(H&E).Then,Observation was made under microscope.
Results:1.Combinated group can establish stable model of acanthamoeba keratitis successfully in Djungarian hamster.This result can be confirmed by 10%potassium hydroxide(KOH) wet mount,corneal cytology,isolation and cultivation of the amoeba,corneal biopsy and histopathological examination.2、In the blank control group、the conditional control group and the experimental group two,no expression of MIP-2 were detected at any observing points.Observation made under light microscope showed no inflammatory cell infiltration too.In the experimental group one,the positive expression of MIP-2 could be detected one day after infection.The level of MIP-2 increased with the time-lapse.The level of MIP-2 reached its peak five days after infection,soon after;its level began to decrease.But,the expression of MIP-2 remained positive seven days after infection.Meanwhile,observation made under light microscope showed there are neutrophil and lymphocyte infiltration at each observing point,especially at 2、3、5d after infection.3.The expression of MIP-2 showed the significant difference between those two experimental groups and the blank control group(P＜0.05).Observation made under light microscope showed that the inflammatory cell infiltration density of those two experimental groups was higher than that of the blank control group 3 and 5 days after infection.The total healing rates of all the observing points in those two experimental groups were 55% and 45%respectively,which were higher than 15%of the blank control group.In the later stage observing points,that is,12d and 18d after infection,the total healing rate in those two experimental groups were 100%and 87.5%respectively,which were higher than 37.5%of the blank control group.
Conclusions:1.By covering ameba-laden contact lenses on the cornea of Djungarian Hamster with preexisting lesions and dropping 20μl suspension of Acanthamoeba (3×10~6 organisms/ml) under it,can establish a successful animal mode of acanthamoeba keratitis.Modeling achievement ratio of this method is high and has a stable course of disease.Although the differences of the affectivity of animal model have no statistical significance between opening the eyelids 48h and 72h after infection,the achievement ratio of opening the eyelids 72h after infection is higher. This method has high achievement ratio ang repeatability.This model can analogue the spontaneous process of human who has infected acanthamoeba keratitis andwould facilitate investigations exploring the pathophysiology,cell biology,genetics, immunology,and therapy of this disease.
2.In effective animal model of acanthamoeba keratitis,neutrophil and lymphocyte infiltration could be detected one day after infection.Subsequently,The quantity of inflammatory cell increased with the time-lapse but began to decrease at seven days after infection.Its developing process was coincided with that of the expression of MIP-2.This result indicated that,as a chemotactic factor,MIP-2 was involved in the immune defence behaviors of acanthamoeba keratitis and played a specific chemotaxis to neutrophil in innate immunity.
3.TNF-αcould induce the up-regulation of MIP-2 gene in the invasion course of acanthamoeba keratitis.Higher expressed MIP-2 could attract more neutrophils involved in the inflammatory reaction,which could accelerate the agglutination of acanthamoeba keratitis without intensifing corneal destruction.
引文
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26. Cavanagh HD, PetrollWM , Alizadeh H, et al. Clinical and diagnostic use of invivo confocal microscopy in patients with comeal disease[J ]. Ophthalmology, 1993,100(10) : 1444.
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30. John T , Desai D , Sahm D. Adherence of Acanthamoeba castellanii cysts and trophozoites to unworn soft contact lenses [ J ]. Am J Ophthalmol 1999 ; 108 :658-664.
31. Ramachandran L , Janakiraman D , Sharma S , Rao GN. Effect of time and washing on the adhesion of Acanthamoeba to extended wear disposable hydrogel contact lenses[ J ]. GLAO J 2005 ; 23 :113-116.
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17 曾庆延,董晓光,史伟云,等．真菌孢子黏附和基质金属蛋白酶在角膜感染 中的作用[J]．中华眼科杂志,2004,40：774—776．
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39 Leher HF , Alizadeh H , Taylor WM , et al. Role of mucosal IgA in the resistance to Acanthamoeba keratitis [ J ]. Invest Ophthalmol Vis Sci1998 ; 39 :2666-2673.
40 D'Aversa G,Stern GA,Driebe WT. Diagnosis and successful medical treatment of A canthamoeba keratitis[J ]. Arch Ophthalmol, 1995, 113 (9) : 1120.
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45 Cavanagh HD, PetrollWM , Alizadeh H, et al. Clinical and diagnostic use of invivo confocal microscopy in patients with corneal disease[J ]. Ophthalmology, 1993,100(10) : 1444.
46 Pfister DR, Cameron JD, Krachmer JH, et al. Confocal microscopy findings of Acanthamoeba keratitis[J ]. Am J Ophthalmol, 1996,121 (2) : 119.
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5.Theodor FH,Jako Biec FA,Juechter KB,et al.The diagnostic value of a ring infiltrate in acanthamoebic keratitis[J].Ophthalmology,1985,92:1471.
6.Silvany RE,Dougherty JM,McCulley JP,et al.The effect of currently available contact lens disinfection systems on Acanthamoeba castellanni and Acanthamoeba polyphaga[J].Ophthalmology,1990,97:286.
7.Niederkorn JY,Ubelake JE,McCulley JP,et al.Susceptibility of corneas from various animal species to in vitro binding and invasion by Acanthamoeba castellanni[J].Invest Ophthalmol Vis Sci,1992,33:104.
8.孙旭光,金秀英.致病性自生生活阿米巴性角膜炎[J].眼科,2003,11:4-6.
9.Kinnear FB.Acanthamoeba pathogenicity for corneal cells[J].J Infect,2004,49:310-316.
10.O'Day DM,Head WS,Csank C,et al.Differences in virulence between two Candida albicans strains in experimental keratitis[J].Invest Ophthalmol Vis Sci,2000,41:1116-1211.
11.曾庆延,董晓光,史伟云,等.真菌孢子黏附和基质金属蛋白酶在角膜感染中的作用[J].中华眼科杂志,2004,40:774-776.
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13.Larkin D F,Easty DL.Experimental Acanthamoeba keratitis:Ⅱ.Immunohisto chemical evaluation [ J ]. Br J Ophthalmol 1991 ; 75 :421-424.
14. Garner A. Pathogenesis of Acanthamoeba keratitis : hypothesis base donahistological analysis of 30 cases [ J ] .Br J Ophthalmol 1993 ; 77 :366-370.
15. van Klink F , Taylor WM ,Alizadeh H , e t al. The role of macrophagesin Acanthamoeba keratitis [ J ]. Invest Ophthalmol Vis Sci 1996 ;37 :1271-1281.
16. van Klink F , Leher H , Jager MJ , e t al. Systemic immune rsponse to Acanthamoeba keratitis in the Chinese hamster [ J ]. Ocul Immunol Inflamm 1997 ; 5 :235-244.
17. McClellan K , Howard K, Mayhew E , Niederkorn J , Alizadeh H. Adaptive immune responses to Acanthamoeba cysts [ J ]. Exp Eye Res 2002 ; 75 :285-293.
18. Niederkorn JY. The role of the innate and adaptive immune responses in Acanthamoeba keratitis [ J ]. Arch Immunol Therap Exp 2002 ; 50 .53-59.
19. Leher HF , Kinoshita K , Alizadeh H , e t al. Impact of oral immunization with Acanthamoeba an tigens on the parasite adhesion and corneal infection [ J ]. Invest Ophthalmol Vis Sci 1998 ; 39 : 2337-2343.
20. Leher HF , Alizadeh H , Taylor WM , et al. Role of mucosal IgA in the resistance to Acanthamoeba keratitis [ J ]. Invest Ophthalmol Vis Sci 1998 ; 39 :2666-2673.
21. Sigrid P.M, Julie Z, Henry M , et al .A synthetic, non-peptide CXCR2 antagonist blocks MIP-2-induced neutrophil migration in mice [ J ]. Immunobiology 2004; 209: 225-233.
22. Li PZ, Sigliti HP, Nagat A, et al. Dynamics of production of MIP-1a, MCP-1 and MIP-2 and potential role of neutralization of these chemokines in the regulation of immune responses during experimental autoimmune neuritis in Lewis rats[ J ]. J Neuroimmunology 1999;98: 168-175.
23. Eva T, Maria LO, Rosario M, et al. Neutralization of macrophage inflammatory protein-2 blocks the febrile response induced by lipopolysaccharide in rats[ J ]. J Thermal Biology 2004;29:413-421.
24. Yong L, Georgia SL, Michael R. Monocyte chemoattractant protein-1 and macrophage inflammatory protein-2 production is inhibited by taurine chloramine in rat C6 glioma cells[ J ] . Immunology L1999;29:9-14.
25. Silvany RE, Dougherty JM , McCulley JP, et al. The effect of currently available contact lens disinfection systems on A canthamoeba castellanni and Acanthamoeba polyphaga [ J ]. Ophthalmology, 1990, 97 (2) : 286.
26. Cavanagh HD, PetrollWM , Alizadeh H, et al. Clinical and diagnostic use of invivo confocal microscopy in patients with comeal disease[J ]. Ophthalmology, 1993,100(10) : 1444.
27. Pfister DR, Cameron JD, Krachmer JH, et al. Confocal microscopy findings of Acanthamoeba keratitis[J ]. Am J Ophthalmol, 1996,121 (2) : 119.
28. Rumelt S, Cohen I, Rehany U. Spontaneous corneal graft ulcerative perforation due to mixed A canthamoeba and herpes simplex keratitis: a clinic opathologic study [J]. Cornea, 2000,19 (2) : 240.
29. Howe DK, Vodk in MH, Novak RJ , et al. Identification of two genetic markers that distinguish pathogenic and nonpathogenic strains of A canthamoeba spp [ J ]. Parasitpl Res, 1997, 83 (3) : 345.
30. John T , Desai D , Sahm D. Adherence of Acanthamoeba castellanii cysts and trophozoites to unworn soft contact lenses [ J ]. Am J Ophthalmol 1999 ; 108 :658-664.
31. Ramachandran L , Janakiraman D , Sharma S , Rao GN. Effect of time and washing on the adhesion of Acanthamoeba to extended wear disposable hydrogel contact lenses[ J ]. GLAO J 2005 ; 23 :113-116.
32. Sharma S , Ramachandran L , Rao GN. Adherence of cysts and trophozoites of Acanthamoeba to unworn rigid gas permeable and soft contact lenses [ J ]. CLAO J 2005; 21 :249-251.
33. Cancrini G , Iori A ,Mancino R . Acanthamoeba adherence to contact lenses , removal by rinsing procedures , and survival to some ophthalmic products [ J ]. Parassitologia 2002 ; 40 :275-278.
34. Schaumberg DA , Snow KK, DanaMR. The epidemic of A canthamoeba keratitis where do stand[J ]. Cornea, 2005, 6(1) : 3.
35. Froum is NA , Mondino BJ , Glasgow BJ. Acanthamoeba keratitis associated with fungal keratitis[J].Am J Ophthalmol,2005,131(4):508.
36.D'A versa G,Stern GA,Driebe W T Jr.Diagnosis and successful medical treatment ofAcanthamoeba keratitis[J].Arch Ophthalmol,2005,113(9):1120.
37.Roters S,Aisenbrey S,Severin M,etal.Painless acanthamoeba keratitis[J].Klin Monatsbl Augenheilkd,2004,218(8):570.
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