【作者】 王维；

【导师】 蒋作君； 沈继龙；

【作者基本信息】 安徽医科大学 ， 流行病与卫生统计学， 2009， 博士

【摘要】 IL-13(Interleukin-13)及其受体IL-13Rα2(IL-13 receptor alpha2)是近年血吸虫病、哮喘等纤维化疾病研究“热点”分子。IL-13主要由活化Th2细胞产生,它有两种亲合力和功能截然不同的受体IL-13Rα1和IL-13Rα2,低亲和力IL-13Rα1可结合IL-4Rα形成异源二聚体、活化IL-13信号通路而发挥生物学效应。相反,IL-13Rα2虽能强力结合IL-13,并且是其细胞内吞所需亚单位,但无信号级连效应,又被称为“诱骗受体(decoy receptor)”。IL-13Rα2或重组sIL-13Rα2-Fc融合蛋白(soluble IL-13Rα2-Fc fusion protein)具有高亲和力拮抗IL-13活性作用,对于某些高表达IL-13受体的肿瘤细胞,利用IL-13运载毒性蛋白IL-13-PE38QQR或高亲和力拮抗剂sIL-13Rα2的受体定点靶向作用,实现对肿瘤细胞特异性“杀死”,目前IL-13Rα2肿瘤基因治疗已进入临床前期实验。IL-13是纤维化细胞外基质重要调节剂,它可直接刺激成纤维细胞产生胶原、导致纤维化,而不影响炎性肉芽肿的形成。目前各种慢性炎性纤维化疾病仍无特效治疗药物,有关IL-13功能和活性及其调节机制的研究将为纤维化疾病干预提供新思路。鼠血吸虫病作为Th2免疫偏移疾病研究模型,IL-13是血吸虫病纤维化最重要介质,曼氏血吸虫病患者及感染动物血清IL-13Rα2水平升高,IL-13Rα2基因敲除鼠( IL-13Rα2-deficient mice, IL-13Rα2-/- )呈程度加重的纤维化,给予注射sIL-13Rα2-Fc后可明显改善纤维化程度,表明纤维化程度加重与IL-13活性增高有关。IL-13Rα2具有下调曼氏血吸虫肉芽肿炎性反应、延长感染鼠寿命等保护性作用。鉴于曼氏血吸虫与日本血吸虫病理机理不尽相同,有关日本血吸虫病IL-13Rα2的研究尚未见报道。更重要的是,Th2细胞因子通过细胞膜上受体实现信号传递,血吸虫感染的机体IL-13应答细胞及其生物学效应尚未阐明。本课题首先以鼠动物模型研究日本血吸虫病感染过程IL-13Rα2表达水平;进一步建立日本血吸虫感染BALB/C鼠巨噬细胞失活模型,通过研究巨噬细胞失活后IL-13Rα2表达变化及肝脏病理改变,探讨肉芽肿IL-13Rα2阳性表达细胞及其免疫病理调节作用。我们的研究内容主要包括二部分:1.日本血吸虫感染诱导小鼠诱骗受体IL-13Rα2表达水平升高采用经腹部皮肤人工感染活尾蚴方法,建立BALB/C鼠日本血吸虫病模型,ELISA法检测血清可溶性IL-13Rα2蛋白动态水平;以IL-13Rα2核苷酸序列(小鼠Accession number NM008356)cDNA为模板设计、合成引物,RT-PCR法扩增肝组织IL-13Rα2基因,获取目的片段;Western blotting检测同期肝组织蛋白表达情况。结果表明:感染鼠血清IL-13Rα2可溶性蛋白水平明显升高,感染后6周达高峰,其后略有下降;PCR法从感染鼠肝脏扩增出胞外区IL-13Rα2 mRNA 800bp片段;Western blotting检测结果显示,肝脏出现特异性IL-13Rα2蛋白条带,相对分子量位于35-49KD之间。因此,日本血吸虫感染诱导机体IL-13Rα2基因转录和蛋白表达增强。2.肉芽肿巨噬细胞特异性增强表达IL-13Rα2及其免疫病理调节作用1)体外实验检测巨噬细胞IL-13Rα2表达水平分离、培养腹腔巨噬细胞,Real time PCR法分析感染鼠巨噬细胞IL-13Rα2 mRNA水平变化(与正常对照组比较);免疫荧光双标法检测IL-13Rα2与CD68共表达情况。结果表明:感染鼠巨噬细胞IL-13Rα2、collagen ?基因水平分别是正常鼠6倍、2.5倍,差异具有显著性(P <0. 01)。细胞免疫荧光染色显示,IL-13Rα2与巨噬细胞标志蛋白CD68呈一致性共表达。体外实验证明:血吸虫感染鼠腹腔巨噬细胞是IL-13Rα2阳性表达细胞。2)体内实验清除IL-13Rα2阳性巨噬细胞具有下调肉芽肿炎性反应和抑制纤维化作用进一步我们给感染血吸虫病BALB/C鼠多次尾静脉注射巨噬细胞清除剂GdCl3,建立巨噬细胞失活日本血吸虫病模型,于感染后42天行门静脉灌流术分离肝脏,对肝组织进行目的基因和组织形态学研究。根据荧光镜下肝肉芽肿ED1免疫阳性信号消失判定模型的成功。其后,三重荧光标记法检测ED1阳性细胞与IL-13Rα2阳性细胞的一致性分布以及TaqManPCR分析基因水平差异情况。结果表明:IL-13Rα2在肉芽肿巨噬细胞特异性增强表达;IL-13Rα2阳性巨噬细胞的消失或失活,小鼠肝脏肉芽肿炎性反应减轻,同期纤维化程度降低。综合我们的研究资料,支持以下结论:日本血吸虫感染诱导机体IL-13Rα2基因转录和蛋白表达增强。肉芽肿巨噬细胞特异性增强表达IL-13Rα2蛋白。清除IL-13Rα2阳性巨噬细胞具有降低肉芽肿炎性反应和减轻纤维化作用,IL-13Rα2+巨噬细胞具有调节肉芽肿免疫病理等重要作用。因此,巨噬细胞增强表达诱骗受体IL-13Rα2可能是血吸虫病肉芽肿及纤维化病理重要调节分子,为Th2相关纤维化疾病基因治疗和侯选细胞靶标的筛选等提供理论依据。更多还原

【Abstract】 Interleukin (IL)-13 and IL-13 receptor (R)α2 appear to play a major role in tissue fibrosis of schistosomiasis and asthma. IL-13 is produced by T helper 2 (Th2) cells. The two binding receptor chains for IL-13 include IL-13Rα1 and IL-13Rα2. When expressed alone, IL-13Rα1 binds IL-13 with low affinity. However, when IL-13Rα1 is coexpressed with IL-4Rα, a high-affinity heterodimerized receptor is formed, regulating the biological activity of IL-13. In contrast, IL-13Rα2, an essential component for binding and internalization of IL-13 but not for IL-13-induced signal transduction, binds IL-13 with high affinity and is known as the decoy receptor. IL-13Rα2 or sIL-13Rα2-Fc fusion protein is highly inhibition of IL-13 activity. To target IL-13 receptor-positive tumor cells, a recombinant fusion IL-13 cytotoxin termed IL-13-PE38QQR or IL-13-PE was developed. It has been shown that IL-13 cytotoxin induces apoptotic cell death in tumor cells. IL-13Rα2 chain is targeted with a receptor-directed cytotoxin of IL-13-PE to induce specific killing in cancer cells. Based on these experimental results, clinical trials of IL-13Rα2 cancer therapy have been initiated.IL-13 is a key regulator of the extracellular matrix. It stimulates collagen production in fibroblasts. Its effects on fibrosis are direct with no effect on granuloma size. Elucidating the mechanisms leading to pathology and fibrosis may lead to more effective strategies for immunological intervention in a variety of chronic diseases. Biological activity and regulation of production and function of IL-13 have become an intensive area of research.IL-13 also plays an important role in schistosomiasis pathogenesis. Serum levels of IL-13Rα2 in both human and mice were upregulated in Schistosoma mansoni (S. mansoni). IL-13Rα2-deficient mice (IL-13Rα2-/-) had a marked exacerbation in hepatic fibrosis. Pathology was prevented when IL-13Rα2-deficient mice were treated with a soluble IL-13Rα2-Fc construct, demonstrating that their exacerbated fibrotic response was due to increased IL-13 activity. IL-13Rα2 down-modulates granulomatous inflammation and prolongs host survival in schistosomiasis. However, much less is known about IL-13Rα2 in S. japonicum, particularly given the difference in the pathology of S. mansoni and S. japonicum. Because Th2 cytokines must bind to their receptors to induce biological responses, the responding cells of IL-13 and the functional activity of IL-13 are unclear. The aim of the present study is to determine in S. japonicum-infected mice: (1) whether the expression of IL-13Rα2 is inducible in a manner similar to that described in S. mansoni studies, and (2) the expression of IL-13Rα2 in macrophages and its effects on immunopathogenesis of schistosomiasis.The main contents are divided into two sections, as follows:1. Enhanced expression of IL-13Rα2 mRNA and protein in S. japonicum miceWe exposed a large number of mice to cercariae and established S. japonicum mouse model. Serum levels of IL-13Rα2 at 6、8、and 12 weeks postinfection were examined with ELISA. Using the Primer Express Software v5.0 with mouse IL-13Rα2 sequence obtained from GenBank (Accession number NM008356), PCR primers were designed and synthesized. IL-13Rα2 mRNA and protein in liver tissues were determined by RT-PCR and immunoblotting analysis, respectively. Result: ELISA revealed that serum levels of soluble IL-13Rα2 were significantly elevated at all time points after infection. The peak levels were at 6 weeks postinfection. 800 bp of IL-13Rα2 cDNA was amplified in whole liver tissues from the infected mice. A specific band of IL-13Rα2 protein between 35-49 KD was detected in the same samples. Hence, the decoy receptor (IL-13Rα2) mRNA and protein in S. japonicum mice were elevated.2. Enhanced expression of IL-13Rα2 in macrophages and its effects in immunopathogenesis of schistosomiasis1) Identification of IL-13Rα2 expression in macrophages in vitroPeritoneal macrophages of infected mice (8 weeks postinfection) or uninfected mice were isolated. RNA was extracted and analyzed using TaqMan PCR. IL-13Rα2 expression in macrophages was performed by the double-labelled fluorescent immunocytochemistry technique with primary antibody of goat anti-mouse IL-13Rα2 antibody and monoclonal anti-CD68 antibody, ED1. The results showed that the level of IL-13Rα2 mRNA was markedly (6-fold) enhanced in infected mice compared to that in uninfected mice. In addition, collagen ? gene was 2.5-fold higher in infected mice relative to healthy controls. As indicated by immunocytochemical staining, IL-13Rα2 was expressed in ED1 positive macrophages. These experiments confirmed the production of IL-13Rα2 by ED1 positive macrophages in schistosomiasis. Hence an enhanced expression of IL-13Rα2 was further demonstrated in primary macrophages of murine schistosomiasis.2) Inactivation of IL-13Rα2 expressing macrophages down-modulates granulomatous inflammation in vivoWe treated mice of schistosomiasis with GdCl3, a reported macrophage depletion reagent. A macrophage-inactivated S. japonicum-infected mouse model was successfully established. Whole liver from each mouse was prepared following perfusion of blood with PBS at 42 days postinfection. Tristain immunofluorescence and TaqMan PCR were used to detect the coexpression of IL-13Rα2 and the macrophage marker (ED1) in liver tissues. These experiments confirmed specifically enhanced expression of IL-13Rα2 in the macrophages of schistosomiasis. Inactivation of IL-13Rα2 expressing macrophages accordingly down-modulates granulomatous inflammation and inhibits fibrosis. Our major findings are summarized below:Expression of the decoy receptor IL-13Rα2 in S. japonicum mice was increased. Specifically enhanced expression of IL-13Rα2 in macrophages of schistosomiasis was confirmed. Inactivation of IL-13Rα2 expressing macrophages down-modulates granulomatous inflammation and inhibits fibrosis. Hence IL-13Rα2 expressed in macrophages may be a critical contributor to the immunopathogenesis of schistosomiasis. Our data highlight the potential importance of IL-13 signaling and antifibrotic therapeutics in Th2-mediated immune diseases.更多还原