CAJ | 학술논문

背景研究证实免疫调节剂CpG ODN对蛋白类过敏原引起的变态反应性结膜炎有防治作用,但其对真菌引发的变态反应性结膜炎是否有类似作用尚不清楚.目的研究局部应用CpG ODN是否能够预防或减轻烟曲霉菌引发的变态反应性结膜炎.方法建立烟曲霉菌变态反应性结膜炎小鼠模型,用CpGODN或对照ODN(GpC ODN)对其进行干预,PBS干预作为阴性对照.实验分为预防实验(模型动物在变态反应原再次激发前给药)和治疗实验(激发后给药),对小鼠眼部临床症状进行评分.取小鼠结膜组织进行组织病理学检查,并计数标本中的炎性细胞.应用实时定量聚合酶链反应(real-time PCR)法动态观察小鼠结膜组织中TLR4 mRNA的变化;将烟曲霉菌免疫原与眼部回流淋巴结和脾脏的细胞进行共培养,流式细胞分析培养细胞中调节性T淋巴细胞的比例和活化状态.结果预防实验中,结膜下注射CpG ODN组与GpC ODN对照组、PBS对照组比较,小鼠眼部过敏症状评分差异均无统计学意义(P＞0.05);CpG ODN组与GpCODN对照组、PBS对照组比较结膜组织中性粒细胞数降低,差异均有统计学意义(21.25±11.59 vs 30.75 ±11.44,21.25±11.59 vs 69.00±9.90;t=5.140,t=3.210,P＜0.01);TLR4 mRNA表达上调.在治疗实验中,CpG ODN组与GpC ODN对照组、模型对照组比较,眼部过敏症状评分降低(t=4.000,t=2.750,P＜0.05);中性粒细胞数显著减少(t=4.870,t=3.829,P＜0.01).CpG ODN治疗组小鼠眼部回流淋巴结CD4+ CD25+细胞以及CD4+ CD25+ CD69+ 细胞所占的比例均显著高于GpC ODN组和PBS对照组(P＜0.05).结论 CpG ODN通过上调结膜TLR4的表达、活化Treg细胞来预防和治疗烟曲霉菌引起的变态反应性结膜炎,预先结膜下注射和点眼均可减轻小鼠眼部过敏症状和晚期结膜炎性细胞的聚集.
배경연구증실면역조절제CpG ODN대단백류과민원인기적변태반응성결막염유방치작용,단기대진균인발적변태반응성결막염시부유유사작용상불청초.목적연구국부응용CpG ODN시부능구예방혹감경연곡매균인발적변태반응성결막염.방법건립연곡매균변태반응성결막염소서모형,용CpGODN혹대조ODN(GpC ODN)대기진행간예,PBS간예작위음성대조.실험분위예방실험(모형동물재변태반응원재차격발전급약)화치료실험(격발후급약),대소서안부림상증상진행평분.취소서결막조직진행조직병이학검사,병계수표본중적염성세포.응용실시정량취합매련반응(real-time PCR)법동태관찰소서결막조직중TLR4 mRNA적변화;장연곡매균면역원여안부회류림파결화비장적세포진행공배양,류식세포분석배양세포중조절성T림파세포적비례화활화상태.결과예방실험중,결막하주사CpG ODN조여GpC ODN대조조、PBS대조조비교,소서안부과민증상평분차이균무통계학의의(P＞0.05);CpG ODN조여GpCODN대조조、PBS대조조비교결막조직중성립세포수강저,차이균유통계학의의(21.25±11.59 vs 30.75 ±11.44,21.25±11.59 vs 69.00±9.90;t=5.140,t=3.210,P＜0.01);TLR4 mRNA표체상조.재치료실험중,CpG ODN조여GpC ODN대조조、모형대조조비교,안부과민증상평분강저(t=4.000,t=2.750,P＜0.05);중성립세포수현저감소(t=4.870,t=3.829,P＜0.01).CpG ODN치료조소서안부회류림파결CD4+ CD25+세포이급CD4+ CD25+ CD69+ 세포소점적비례균현저고우GpC ODN조화PBS대조조(P＜0.05).결론 CpG ODN통과상조결막TLR4적표체、활화Treg세포래예방화치료연곡매균인기적변태반응성결막염,예선결막하주사화점안균가감경소서안부과민증상화만기결막염성세포적취집.
Background Researches demonstrated that CpG ODN,a immunostimulatory sequences,has preventing and treating effect on allergic conjunctivitis caused by protein allergen.However,its effect on allergic conjunctivitis caused by fungal allergen is unclear. Objective This study aimed to investigate into whether the Th1-Th2 switching immunostimulatory CpG ODN could reverse the response in the murine allergic conjunctivitis model caused by aspergillus fumigatus. Methods A mixture of spores and hyphae of aspergillus fumigatus strain was used to induce allergic conjunctivitis in male BALB/C mice aged 6-8 weeks.This experiment was designed into preventive or therapeutical treatment program.Under both settings,allergic conjunctivitis of the animals were treated with CpG ODN,nonstimulatory GpC ODN or PBS.After the last challenge with the allergen,the clinical symptoms of the animals were scored based on the criteria of Magone.The animals were sacrificed and the histopathological examination of conjunctiva was performed.Expression of TLR4 mRNA in conjunctiva was analyzed by real-time PCR assay.The responsiveness and populations of lymphocytes in spleen and draining lymph nodes were analyzed by flow cytometry.The use complied with the Standard of Association for Research in Vision and Ophthalmology. Results In the prevention mode.CpG ODN decreased subconjunctival infiltration compared with GpC ODN and PBS groups with the average neutrophil count index(21.25 ±11.59/section,30.75 ±11.44 section and 69.00±9.90/section,respectively).Expression of TLR4 mRNA was up-regulated significantly by CpG ODN.The clinical scores for CpG ODN group were insignificantly lower than those in GpC ODN group and PBS group(P＞0.05).In the therapeutic mode,compared with GpC ODN and PBS groups,the allergic symptom score in CpG ODN group manifested significantly lower(t=4.000.t=2.750,P＜0.01)and showed fewer cellular infiltration(t=4.870,t=3.829,P＜0.01)and higher expression of TLR4 mRNA(P＜0.01).In cultured splenic and draining lymph node cells,increased percentages of CD4+ CD25+ and CD4+ CD25+ CD69+ in CpG ODN group were observed compared with control groups(|P＜0.05). Conclusion CpG ODN can relieve aspergillus fumigatus-induced allergic conjunctivitis via either subconjunctival injection or topical application by upregulating expression of TLR4 and activating Treg lymphocytes.