免疫刺激剂CpGODN防治烟曲霉菌引起的变态反应性结膜炎的实验研究

Effects of immunostimulatory CpG ODN on experimental allergic conjunctivitis caused by aspergillus fumigatus

背景研究证实免疫调节剂CpGODN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CpGODN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（GpCODN）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-timePCR）法动态观察小鼠结膜组织中TLR4mRNA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CpGODN组与GpCODN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）；CpGODN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±11．59vs30．75-4-_11．44，21．25±11．59vs69．00±9．90；t=5．140，t=3．210，P〈0．01）；TLR4mRNA表达上调。在治疗实验中，CpGODN组与GpCODN对照组、模型对照组比较，跟部过敏症状评分降低（t=4．000，t=2．750，P〈O．05）；中性粒细胞数显著减少（t=4．870，t=3．829，P〈0．01）。CpGODN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GpCODN组和PBS对照组（P〈0．05）。结论CpGODN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。

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 Thl-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 ofthe 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 CD^4＋ 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.