摘要
目的
探讨β-葡聚糖特异性识别受体Dectin-1对茄病镰刀菌及烟曲霉菌的识别及其在正常角膜组织和真菌感染角膜组织中的表达及特点,并初步探讨其在真菌性角膜炎中作用的分子机制。
方法
第一部分:真菌刺激对腹腔巨噬细胞Dectin-1表达的影响
1.小鼠腹腔巨噬细胞的培养及鉴定C57BL/6小鼠腹腔注射巯基乙酸钠肉汤(4%),注射后第3天,收集腹腔巨噬细胞,体外培养24hr,以F4/80抗体及CD68抗体进行鉴定并利用流式细胞术检测细胞纯度。
2.真菌刺激对巨噬细胞模式识别受体Dectin-1表达的影响利用纯化的β-葡聚糖Laminarin、茄病镰刀菌及烟曲霉菌孢子刺激培养的巨澨细胞,免疫荧光技术及RTQ-PCR检测Dectin-1的表达变化。
第二部分:Dectin-1在小鼠真菌性角膜炎模型中的表达及特点
角膜基质注射法建立C57BL/6小鼠茄病镰刀菌及烟曲霉菌角膜感染模型,角膜基质注射生理盐水小鼠作为阴性对照组,末处理的正常角膜为空白对照组。感染后1、2、3、5、7天取各组角膜行RTQ-PCR检测,观察Dectin-1的表达情况。角膜组织病理学方法观察角膜内炎症细胞的侵润,探讨侵润细胞与Dectin-1表达量的关系。
第三部分:Dectin-1在真菌性角膜炎中作用分子机制的初步研究
1.RTQ-PCR检测真菌刺激巨噬细胞及真菌感染的角膜中CARD9的表达;
2.RTQ-PCR检测主要细胞因子及趋化因子IL-1β、IL-2、IL-6、MIP-2、IL-10、IL-12、IL-17、MCP-1、TNF-α、CXCL1/KC在Laminarin刺激细胞及真菌感染角膜中的表达;
3.Dectin-1多克隆抗体阻断体外培养的巨噬细胞表面的Dectin-1,再利用茄病镰刀菌及烟曲霉菌刺激细胞,检测Dectin-1抗体阻断前后细胞培养上清液中IL-1β的含量。
结果
第一部分:真菌刺激对腹腔巨噬细胞Dectin-1表达的影响
小鼠腹腔注射巯基乙酸钠肉汤3天后,每只小鼠腹腔大约可收集1×107个细胞。体外培养的巨噬细胞贴壁生长,呈圆形、长形及不规则三角形等,可见突触。F4/80抗体免疫荧光染色后,流式细胞术检测,可发现F4/80阳性细胞约占86%,说明此方法分离的腹腔巨噬细胞纯度较高,可以进行相关的功能性研究。
真菌刺激后巨噬细胞表面的Dectin-1表达上调。Laminarin与培养的巨噬细胞共同孵育12hr后,RTQ-PCR结果显示Dectin-1的表达并未升高(P=0.261)。而茄病镰刀菌孢子和烟曲霉菌孢子与细胞以1:10的比例共同孵育12hr后,受真菌刺激的细胞Dectin-1的表达则明显上调(P=0.000 & P=0.009)。
第二部分:Dectin-1在小鼠真菌性角膜炎模型中的表达及特点
RTQ-PCR结果及Dectin-1免疫荧光染色的结果均显示,正常及阴性对照角膜组织中没有Decitin-1的表达。但是在真菌感染的角膜中,可以检测到Dectin-1的表达。Dectin-1的表达量与炎症细胞侵润程度紧密相关,在感染后的第2、3天,感染角膜组织内炎症细胞侵润最为明显,同时Dectin-1的表达水平也最高。随着病程的进展,炎症细胞的数量减少,Dectin-1的表达量也随之下降。对侵润的炎症细胞进行F4/80和Gr-1免疫荧光染色,结果则显示,侵润的巨噬细胞和中性粒细胞表达Dectin-1。
第三部分:Dectin-1在真菌性角膜炎中作用分子机制的初步研究
真菌感染的角膜中,CARD9的表达明显上调,其上调趋势与Dectin-1一致。腹腔巨噬细胞表面Dectin-1对p-葡聚糖的识别和清除过程中,可介导IL-1β、MIP-2、IL-12、CXCL1/KC、MCP-1等多种细胞因子的产生,上调最明显的为IL-1β、IL-12和IL-6。在两种丝状真菌的角膜炎模型中,也均可观察到上述细胞因子的表达明显上调,其中表达上调最显著的细胞因子为IL-1β、MIP-2、CXCL1/KC和IL-12。其上调及下调的变化趋势与Dectin-1及CARD9变化趋势一致。Dectin-1抗体中和实验显示:中和抗体阻断Dectin-1后,烟曲霉菌孢子与细胞共孵育24hr后,IL-1β的表达量下降(P=0.016)。
结论
1、茄病镰刀菌和烟曲霉菌可激活腹腔巨噬细胞表面的Dectin-1。
2、正常的小鼠角膜组织不表达Dectin-1,但受真菌感染的角膜组织有Dectin-1的表达。
3、FK小鼠角膜中,Dectin-1的表达量与炎症细胞的侵润程度相关。
4、CARD9在FK中表达上调,说明其介导的信号通路可能参与了FK的发病过程。
5、Dectin-1识别β-葡萄糖后,可诱导IL-1β、MIP-2、KC、IL-12等细胞因子的产生;茄病镰刀菌与烟曲霉菌发病过程中细胞因子的产生及表达变化趋势致。
6、采用特异性的Dectin-1多克隆抗体中和巨噬细胞表面的Dectin-1,可以部分阻断IL-1β的表达。
7、Dectin-1介导的信号通路参与了FK的病理过程,可能是通过细胞因子的产生介导抗真菌免疫保护。因此,以Dectin-1或真菌细胞壁β-葡聚糖作为控制靶点,有望找到治疗及调控FK的新方法。但其具体分子机制还需要进一步的研究。
Objective To identify the response and the underlying response pathways of Dectin-1 on macrophages to Fusarium solani and Aspergillus fumigatus. And then we sought to determine whether Dectin-1 plays a similar role in beta-glucan-induced activation in fungal keratitis.
Methods Peritoneal macrophages isolated after intraperitoneal injection of sodium thioglycollate were co-cultured with Laminarin, a purified beta-glucan and spores of Fusarium solani and Aspergillus fumigatus for 12 hours. The expression level of Dectin-1 and several cytokines was evaluated by immunofluorescence and real-time quantitative polymerase chain reaction (RTQ-PCR). Then mouse fungal keratitis model were establish by injection of inoculation fungal spores substromally. corneal lesions and inflammatory responses were observed by slit-lamp and histopathology at one, two, three, five and seven days post-infection. RTQ-PCR was used to detect the levels of Dectin-1,CARD9, CXCL1/KC and interleukin (IL)-1β,IL-2,1L-6, macrophage inflammatory protein-2 (MIP-2),IL-10,1L-12,1L-17, tumor necrosis factor a (TNF-a) and monocyte chemotactic protein-1 (MCP-1). For the intervention experiment with neutralizing antibody, macrophages were elicited and cultured for 24 hours and then co-cultured with Dectin-1 antibody (25μg/ml) for 1 hour at 37℃before stimulated with Laminarin and spores. The level of IL-1βwas measured by ELISA.
Results Expression of Dectin-1 was upregulated significantly on macrophages stimulated by spores of Fusarium solani and Aspergillus fumigatus, but not by Laminarin(P=0.261). Dectin-1 was not detected in normal corneas of C57BL/6 mouse. But it was detected in infected corneas from the first day post-infection and the mRNA level was highest on the second and third days. Dectin-1 was expressed on the surface of macrophages (F4/80+) and neutrophilic granulocytes (Gr-1+) presence in the infected cornea. CARD9, a key transducer of Dectin-1 signalling, was also upregulated at the same time with Dectin-1 on macrophages and the infected corneas. IL-17 was not detected on macrophages with or without stimulation by Laminarin. Expression of the other examined cytokines were upregulated on macrophages stimulated by Laminarin except TNF-a (P=0.096). The cytokines which were upregulated most significantly were IL-1β、IL-6 and IL-12. All of the examined proinflammatory cytokines and inflammatory factors were produced in detectable in murine fungal keratitis. And,the predominances were IL-1β、MIP-2、CXCL1/KCand IL-12. The produced cytokines were similar in fungal keratitis caused by the two filamentous fungi. Upon administration of Dectin-1 polyclonal antibodies on macrophages, the decrease in mRNA level of IL-1βwas apparent (p=0.016).
Conclusion Fusarium solani and Aspergillus fumigatus up-regulate the expression of Dectin-1 in mouse peritoneal macrophages. Dectin-1 could be detected in corneas infected by Fusarium solani and Aspergillus fumigatus but not in normal and mock-infected corneas. CARD9 is activated in FK and in vivo production of IL-1β, MIP-2, CXCL1/KC and IL-12 was significantly increased as compared to control groups. Dectin-1 polyclonal antibodies can reduce IL-1βproduced by macrophages. Thus, Dectin-1 may play an important role in FK and further study is still required about the molecular mechanisms.
引文
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