中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2014年
10期
870-875
,共6页
渠晓黎%赵桂秋%许正杰%高昂%王楠%刘莹%林静
渠曉黎%趙桂鞦%許正傑%高昂%王楠%劉瑩%林靜
거효려%조계추%허정걸%고앙%왕남%류형%림정
烟曲霉菌%眼部感染,真菌%角膜炎%核苷酸结合寡聚域%天然免疫%大鼠
煙麯黴菌%眼部感染,真菌%角膜炎%覈苷痠結閤寡聚域%天然免疫%大鼠
연곡매균%안부감염,진균%각막염%핵감산결합과취역%천연면역%대서
Aspergillus fumigatus%Eye Infection,Fungal%Keratitis%Nucleotide binding oligomerization domain%Innate immunity%Rat
背景 核苷酸结合寡聚域(NOD)样受体(NLRs)在天然免疫过程中发挥重要作用,但其在真菌性角膜炎发生和发展过程中的作用研究少见. 目的 研究NOD2在大鼠烟曲霉菌性角膜炎(AFK)角膜组织中的表达及作用.方法 72只成年清洁级Wistar大鼠按照随机数字表法随机分为正常对照组、单纯角膜上皮损伤组和AFK模型组,均以右眼作为实验眼.正常对照组12只大鼠中选取6只仅刮取角膜上皮用于逆转录PCR(RT-PCR)实验,另6只大鼠制备角膜标本用于组织病理学检查、免疫组织化学和免疫荧光染色实验.单纯角膜上皮损伤组共30只大鼠,仅刮除中央角膜上皮,AFK模型组大鼠共30只,刮除角膜上皮后接种烟曲霉菌标准株.分别于实验后4、8、16、24 h选取各组6只大鼠刮取角膜上皮,采用RT-PCR法检测角膜中NOD2mRNA的表达,其他6只于实验后24 h制备角膜标本,采用免疫组织化学法和免疫荧光染色法检测角膜中NOD2蛋白的表达,并将各组检测结果进行比较.实验后4、8、16、24 h分别摘除各组大鼠右眼眼球,制作角膜标本进行常规组织病理学检查.结果 裂隙灯显微镜下观察各组大鼠各时间点的角膜情况,均造模成功.RT-PCR结果显示,正常对照组角膜仅有微量NOD2 mRNA的表达,单纯角膜上皮损伤组和AFK模型组大鼠角膜中NOD2 mRNA的相对表达量明显增加,且在实验4、8、16、24 h,AFK模型组大鼠角膜中NOD2 mRNA的相对表达量均明显高于单纯角膜上皮损伤组,差异均有统计学意义(t=-0.409、-0.439、-0.534、-0.618,均P=0.000).大鼠角膜组织病理学检查显示,正常对照组大鼠角膜结构完整;单纯角膜上皮损伤组可见到少部分角膜上皮缺损、前弹力层皱褶及角膜轻度水肿,有少量中性粒细胞浸润;AFK模型组大鼠可见角膜溃疡,角膜水肿增厚,浅基质层可见大量中性粒细胞浸润.免疫组织化学染色和免疫荧光染色结果均显示,正常对照组大鼠角膜上皮和内皮层仅有微弱NOD2蛋白的表达,单纯角膜上皮损伤组大鼠NOD2蛋白表达稍增强,AFK模型组大鼠NOD2蛋白表达明显增强.3个组大鼠角膜中NOD2蛋白表达量(A值)分别为0.045±0.005、0.050±0.005和0.092±0.006,其中AFK模型组大鼠NOD2蛋白表达量明显高于单纯角膜上皮损伤组,差异有统计学意义(t=0.042,P=0.000). 结论 NOD2参与了大鼠AFK的早期病变过程,可能在角膜抗真菌感染的天然免疫阶段中发挥一定的作用.
揹景 覈苷痠結閤寡聚域(NOD)樣受體(NLRs)在天然免疫過程中髮揮重要作用,但其在真菌性角膜炎髮生和髮展過程中的作用研究少見. 目的 研究NOD2在大鼠煙麯黴菌性角膜炎(AFK)角膜組織中的錶達及作用.方法 72隻成年清潔級Wistar大鼠按照隨機數字錶法隨機分為正常對照組、單純角膜上皮損傷組和AFK模型組,均以右眼作為實驗眼.正常對照組12隻大鼠中選取6隻僅颳取角膜上皮用于逆轉錄PCR(RT-PCR)實驗,另6隻大鼠製備角膜標本用于組織病理學檢查、免疫組織化學和免疫熒光染色實驗.單純角膜上皮損傷組共30隻大鼠,僅颳除中央角膜上皮,AFK模型組大鼠共30隻,颳除角膜上皮後接種煙麯黴菌標準株.分彆于實驗後4、8、16、24 h選取各組6隻大鼠颳取角膜上皮,採用RT-PCR法檢測角膜中NOD2mRNA的錶達,其他6隻于實驗後24 h製備角膜標本,採用免疫組織化學法和免疫熒光染色法檢測角膜中NOD2蛋白的錶達,併將各組檢測結果進行比較.實驗後4、8、16、24 h分彆摘除各組大鼠右眼眼毬,製作角膜標本進行常規組織病理學檢查.結果 裂隙燈顯微鏡下觀察各組大鼠各時間點的角膜情況,均造模成功.RT-PCR結果顯示,正常對照組角膜僅有微量NOD2 mRNA的錶達,單純角膜上皮損傷組和AFK模型組大鼠角膜中NOD2 mRNA的相對錶達量明顯增加,且在實驗4、8、16、24 h,AFK模型組大鼠角膜中NOD2 mRNA的相對錶達量均明顯高于單純角膜上皮損傷組,差異均有統計學意義(t=-0.409、-0.439、-0.534、-0.618,均P=0.000).大鼠角膜組織病理學檢查顯示,正常對照組大鼠角膜結構完整;單純角膜上皮損傷組可見到少部分角膜上皮缺損、前彈力層皺褶及角膜輕度水腫,有少量中性粒細胞浸潤;AFK模型組大鼠可見角膜潰瘍,角膜水腫增厚,淺基質層可見大量中性粒細胞浸潤.免疫組織化學染色和免疫熒光染色結果均顯示,正常對照組大鼠角膜上皮和內皮層僅有微弱NOD2蛋白的錶達,單純角膜上皮損傷組大鼠NOD2蛋白錶達稍增彊,AFK模型組大鼠NOD2蛋白錶達明顯增彊.3箇組大鼠角膜中NOD2蛋白錶達量(A值)分彆為0.045±0.005、0.050±0.005和0.092±0.006,其中AFK模型組大鼠NOD2蛋白錶達量明顯高于單純角膜上皮損傷組,差異有統計學意義(t=0.042,P=0.000). 結論 NOD2參與瞭大鼠AFK的早期病變過程,可能在角膜抗真菌感染的天然免疫階段中髮揮一定的作用.
배경 핵감산결합과취역(NOD)양수체(NLRs)재천연면역과정중발휘중요작용,단기재진균성각막염발생화발전과정중적작용연구소견. 목적 연구NOD2재대서연곡매균성각막염(AFK)각막조직중적표체급작용.방법 72지성년청길급Wistar대서안조수궤수자표법수궤분위정상대조조、단순각막상피손상조화AFK모형조,균이우안작위실험안.정상대조조12지대서중선취6지부괄취각막상피용우역전록PCR(RT-PCR)실험,령6지대서제비각막표본용우조직병이학검사、면역조직화학화면역형광염색실험.단순각막상피손상조공30지대서,부괄제중앙각막상피,AFK모형조대서공30지,괄제각막상피후접충연곡매균표준주.분별우실험후4、8、16、24 h선취각조6지대서괄취각막상피,채용RT-PCR법검측각막중NOD2mRNA적표체,기타6지우실험후24 h제비각막표본,채용면역조직화학법화면역형광염색법검측각막중NOD2단백적표체,병장각조검측결과진행비교.실험후4、8、16、24 h분별적제각조대서우안안구,제작각막표본진행상규조직병이학검사.결과 렬극등현미경하관찰각조대서각시간점적각막정황,균조모성공.RT-PCR결과현시,정상대조조각막부유미량NOD2 mRNA적표체,단순각막상피손상조화AFK모형조대서각막중NOD2 mRNA적상대표체량명현증가,차재실험4、8、16、24 h,AFK모형조대서각막중NOD2 mRNA적상대표체량균명현고우단순각막상피손상조,차이균유통계학의의(t=-0.409、-0.439、-0.534、-0.618,균P=0.000).대서각막조직병이학검사현시,정상대조조대서각막결구완정;단순각막상피손상조가견도소부분각막상피결손、전탄력층추습급각막경도수종,유소량중성립세포침윤;AFK모형조대서가견각막궤양,각막수종증후,천기질층가견대량중성립세포침윤.면역조직화학염색화면역형광염색결과균현시,정상대조조대서각막상피화내피층부유미약NOD2단백적표체,단순각막상피손상조대서NOD2단백표체초증강,AFK모형조대서NOD2단백표체명현증강.3개조대서각막중NOD2단백표체량(A치)분별위0.045±0.005、0.050±0.005화0.092±0.006,기중AFK모형조대서NOD2단백표체량명현고우단순각막상피손상조,차이유통계학의의(t=0.042,P=0.000). 결론 NOD2삼여료대서AFK적조기병변과정,가능재각막항진균감염적천연면역계단중발휘일정적작용.
Background Studies have determined that nucleotide binding oligomerization domain 2 (NOD2) plays a key role in innate immune response.However,whether NOD2 participates in the nature defense of fungal keratitis is unclear.Objective This study was to investigate the expression and significance of NOD2 on cornea in the initial of Aspergillus fumigatus keratitis (AFK) in rats.Methods Seventy-two adult clean Wistar rats were randomized into the normal control group,only corneal epithelial scraped group and AFK model group,and the AFK models were established by incubating Aspergillus fumigatus to cornea after corneal epithelium was scraped.All the operations were performed in the right eyes of rats.Reverse transcription PCR (RT-PCR) was carried out to detect the expression of NOD2 mRNA in corneal epithelium 4,8,16,24 hours after operation.Twenty-four hours after operation,the expression of NOD2 protein in rat corneas was examined by immunochemistry and immnunofluorescence technology.Also,the rat corneas were obtained for regular histopathological examination.The use and care of the animals complied with Institutional Animal Care and Use Committee Guidebook by NIH.Results All the models were made successfully.RT-PCR revealed that a fewer NOD2 mRNA were expressed on cornea in the normal control group,but the expressing levels of NOD2 mRNA were increased in the only corneal epithelial scraped group and AFK model group.Compared with only corneal epithelial scraped group,the elevated values of NOD2 mRNA expression in the AFK model group were statistically significant at 4,8,16 and 24 hours after operation (t =-0.409,-0.439,-0.534,-0.618,all at P=0.000).The histopathological examination displayed that the cornel tissue had intact structure in the normal control group,and partly corneal epithelial deficiency,slight corneal swelling and fewer neutrophil granulocytes were seen in the only corneal epithelial scraped group.However,corneal ulcer,severe corneal edema and a lot of neutrophil granulocytes were exhibited in the AFK model group.Immunochemistry and immnunofluorescence staining evidenced that weaker expression of NOD2 was visualized in the corneal epithelial and endothelial layers,and obviously enhanced staining was seen in the AFK model group.The expressing levels (absorbancy) were 0.045 ± 0.005,0.050 ± 0.005 and 0.092 ± 0.006 in the normal control group,only corneal epithelial scraped group and AFK model group,respectively,showing a significant increase in the AFK model group compared with the only corneal epithelial scraped group (t =0.042,P =0.000).Conclusions Expression of NOD2 is upregulated in the corneas with AFK,suggesting that NOD2 participates the natural defense in the initial of fungal keratitis.NOD2 may play an important role in the process of anti-fungal innate immune response in cornea.
