摘要
舍格伦综合征是一种可以引起外分泌腺分泌功能障碍的慢性系统性自身免疫疾病。根据临床特征可以分为两种类型:原发性舍格伦综合征和继发性舍格伦综合征。原发性舍格伦综合征特点是干燥性角结膜炎和口干症;继发性舍格伦综合征是原发性的症状伴发其他部位的自身免疫疾病,例如风湿性关节炎、系统性红斑狼疮和硬皮病等。舍格伦综合征的发病率为0.5-3%,女性中多见(是男性患者人数的9倍)。舍格伦综合征的发病机制至今尚不清楚,多种因素与其发病相关,例如内分泌因素、遗传因素、神经系统紊乱等,但最关键的致病因素是免疫系统的异常。
IL-21是一种具有多种功能的I型细胞因子,在淋巴细胞的发育、增殖和分化过程中,尤其在抗体产生和浆细胞成熟过程中发挥关键作用。同时,很多研究也证明IL-21参与了多种自身免疫性疾病的发展过程,例如感染性肠病、风湿性关节炎、糖尿病、系统性红斑狼疮等,所以IL-21也被当做自身免疫性疾病治疗中的一种关键因子和治疗靶位。但IL-21在舍格伦综合征的作用尚不清楚,IL-21在舍格伦综合征发病中的作用和具体机制值得深入研究。
因此,为了搞清IL-21在舍格伦综合征发病过程中的作用及其机制,我们选用了常用的舍格伦综合征模型动物NOD小鼠作为研究对象,通过在NOD小鼠中阻断IL-21信号通路,观察IL-21对NOD小鼠舍格伦综合征样症状的影响,并进一步优化NOD小鼠体内IL-21的干扰手段后,对IL-21在舍格伦综合征发病过程中的具体机制进行深入的研究。
第一部分:在NOD小鼠体内阻断IL-21信号对舍格伦综合征发病的影响
目的:使用融合蛋白IL-21R.Fc竞争性阻断IL-21与IL-21受体的结合,抑制NOD小鼠体内IL-21信号通路,检测NOD小鼠舍格伦综合征症状的变化,从而研究IL-21对舍格伦综合征发病的影响。
方法:利用分子克隆技术以小鼠cDNA为模版分别PCR扩增IL-21受体胞外区片段和小鼠IgG2a的Fc片段,其中Fc片段的235、3]8、320和322为氨基酸进行诱导定点突变,以减少Fc受体结合活性和补体激活能力。然后将IL-21受体胞外区片段和Fc段基因插入质粒pcDNA3.1+构建成融合蛋白IL-21R.Fc的真核表达质粒pcDNA3.1-IL-21R.Fc。(?)勾建好的质粒用脂质体瞬时转染CHO细胞进行表达,收集培养上清和细胞裂解液利用亲和层析法对融合蛋白进行纯化,并进行SDS-PAGE电泳检验。将纯化的融合蛋白IL-21R.Fc通过尾静脉注射作用于NOD小鼠体内,定期检测唾液流速和颌下腺HE染色病理切片,对NOD小鼠的唾液分泌功能和颌下腺炎症浸润情况进行评价。
结果:质粒pcDNA3.1-IL-21R.Fc构建成功,转染CHO细胞后成功表达并纯化得到融合蛋白IL-21R.Fc。通过融合蛋白IL-21R.Fc在NOD小鼠体内对IL-21信号的抑制作用,缓解了NOD小鼠的舍格伦综合征症状。唾液分泌功能障碍的进展减慢,颌下腺中的淋巴细胞浸润也得到了缓解。
结论:IL-21信号在NOD小鼠的舍格伦综合征发病过程中发挥了重要的致病作用。对IL-21信号的阻断和抑制可能作为舍格伦综合征的一种有效治疗手段。
第二部分:在NOD小鼠颌下腺局部抑制IL-21表达对舍格伦综合征的影响机制
目的:使用表达IL-21shRNA的慢病毒表达载体通过颌下腺导管逆行灌注,在NOD小鼠颌下腺中长期抑制IL-21的表达,观察对NOD小鼠舍格伦综合征的影响,并对IL-21的作用机制进行研究分析。
方法:通过IL-21shRNA的慢病毒载体逆行灌注NOD小鼠颌下腺,检查慢病毒对IL-21表达的抑制效果,定期检测唾液流速和颌下腺淋巴浸润情况。利用实时定量PCR对辅助T淋巴细胞相关细胞因子mRNA水平进行检测,研究IL-21抑制对NOD小鼠颌下腺中各种细胞因子的影响。利用流式细胞术和免疫组织化学法对NOD细胞中的CD4+CXCR5+Tfh细胞进行检测,观察IL-21抑制对Tfh细胞的影响。
结果:IL-21shRNA的慢病毒表达载体明显抑制了IL-21在NOD小鼠颌下腺中的表达,并缓解了NOD小鼠的唾液分泌功能障碍和颌下腺淋巴浸润的进展。通过对NOD小鼠颌下腺中多种细胞因子的表达进行的实时定量PCR检测,发现IL-21的抑制对Tfh细胞相关的CXCR5和BCL-6有明显的抑制作用。NOD小鼠颌下腺中CD4+CXCR5+Tfh细胞占CD4+T淋巴细胞的比率在IL-21的表达受到抑制后得到明显降低,免疫组织化学法也发现颌下腺中CXCR5+细胞随着IL-21的抑制而减少。
结论:在NOD小鼠颌下腺中局部抑制IL-21缓解了颌下腺的分泌功能障碍和淋巴细胞浸润的进展,延缓了舍格伦综合征在NOD小鼠颌下腺中的发展。IL-21的抑制主要通过影响Tfh细胞的分化发育,从而影响了生发中心的形成和自身反应性的浆细胞的成熟。
综上所述,本课题证实了IL-21在舍格伦综合征发病过程中的重要致病作用,并且.验证了局部抑制IL-21在治疗舍格伦综合征中的有效性。根据实验数据我们发现IL-21通过影响Tfh细胞的分化发育,影响了颌下腺中生发中心的形成和自身反应性的浆细胞的成熟,从而导致了舍格伦综合征的发展。进一步探索IL-21的干预手段对舍格伦综合征的治疗有着重要的意义。
Sjogren's syndrome (SjS) is considered an autoimmune disease in which the immune system targets the exocrine glands and leads to the loss of secretory function. SjS is classified as either primary SjS or secondary SjS. Primary SjS is a chronic autoimmune attack involving both lacrimal and salivary glands in the absence of other autoimmune diseases, whereas secondary SjS is an autoimmune attack against the lacrimal and/or salivary glands in the presence of other autoimmune diseases, most often rheumatoid arthritis, systemic lupus erythmatosus (SLE), or scleroderma. SjS is one of the three most common autoimmune diseases, along with SLE and systemic sclerosis. SjS affects0.5-3%of the population, and predominates in females with a ratio of9:1versus males, suggesting hormonal involvement. The pathogenesis of SjS is complicated with many respects remaining elusive.
IL-21is a pleiotropic type Ⅰ cytokine and plays critical roles in lymphocyte development, proliferation, and differentiation, especially in antibody production and the maturation of B cells into primary antibody-producing plasma cells. Accumulated research of IL-21has associated IL-21with the development of many autoimmune diseases, such as inflammatory bowel disease, rheumatoid arthritis, diabetes and systemic lupus erythematosus. So IL-21is considered as a key factor and therapeutic agent in the treatment of autoimmune diseases. The role of IL-21in the development of Sjogren's syndrome remained elusive. Many studies have shown that the level of serum IL-21in patients with SjS is higher than that in healthy control, and minor salivary glands biopsy specimens revealed positive staining for both IL-21and IL-21receptor within lymphocytic foci and periductal area whereas only minimal expression was seen in samples of control. However, the role of IL-21in the pathogenesis of SjS remains elusive and its mechanism is not clear so far.
In order to figure out the role of IL-21in SjS, SjS model animal nonobese diabetic (NOD) mouse was used in our study. We blocked IL-21pathway in NOD mice to evaluate the role of IL-21in the development of SjS and figure out the mechanism.
Part Ⅰ. The role of IL-21blockage played in development of SjS of NOD mice
Object:To evaluate the role of IL-21played in development of SjS, fusion protein IL-21R.Fc was used to block IL-21signal in NOD mice and the changes of SjS symptoms were monitored.
Methods:The extracellular domain of IL-21R and Fc domain of IgG2a of mice were amplified from mouse cDNA following the molecular cloning methods. The235,318,320and322aa of Fc domain were mutated to minimize Fc binding and complement fixation. DNA segments of murine IL-21R extracellular domain and mutated IgG2a Fc domain were connected and cloned into plasmid pcDNA3.1+to construct plasmid pcDNA3.1-IL-21R.Fc. Plasmid pcDNA3.1-IL-21R.Fc was purified and transfected into CHO cells with Lipofectamine2000. IL-21R.Fc fusion protein was purified through Protein A affinity columns with CHO cell lysates and conditioned medium. The protein was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. NOD mice were administered intravenuously in tail with purified IL-21R.Fc. Saliva flow rate and lymphocytic infiltration in submandibular glands of NOD mice were evaluated every4weeks.
Result:Plasmid pcDNA3.1-IL-21R.Fc was constructed successfully, and fusion protein IL-21R.Fc was purified. Blockage of IL-21signal by IL-21R.Fc in NOD mice alleviated symptoms of SjS. The development of secretory dysfunction and lymphocytic infiltration were retarded in submandibular glands.
Conclusions:IL-21played a critical role in the development of SjS in NOD mice. IL-21pathway could be used as a promising therapeutic target in the treatment of SjS.
Part Ⅱ. The role of local suppression of IL-21in submandibular glands of NOD mice played in development of SjS and mechanism research
Object:The aim of this study was to verify the validity of IL-21local suppression in submandibular glands of preventing the development of SjS in non-obese diabetic (NOD) mice and try to figure out the mechanism.
Methods:IL-21levels in submandibular glands were suppressed by ductal cannulation of IL-21shRNA lentivirus. Then, saliva flow rates and histopathologic changes of submandibular glands were measured to assess the severity of disease development. Real-time PCR, flow cytometry, and immunohistochemistry were used to detect the changes of T helper cells and related cytokines.
Result:The reduction in SFRs in NOD mice was significantly alleviated from9to17weeks of age along with the suppression of IL-21in submandibular glands. Lymphocytic infiltration was also milder than control NOD mice. Moreover, the lower level of IL-21led to the down-regulation of follicular helper T (Tfh) cells.
Conclusions:Local suppression of IL-21alleviated secretory disfunction and disrupted lymphocyte infiltration in SMG of NOD mice and thus retarded the development of SjS-like symptoms of NOD mice. Suppression of IL-21might interfere with the development of GC and maturation of B cells through Tfh cells.
In summary, our study proved the important role of IL-21in the development of SjS, and found out that local suppression of IL-21is effective in the treatment of SjS. IL-21could contribute to the development of SjS through Tfh cells to improve the formation of GC and the maturation of B cells in submandibular glands. Further research is needed on IL-21that is a promising target in the treatment of SjS.
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