腺病毒载体介导的“睡美人”转座子与IL-10共表达对干燥综合征动物模型NOD小鼠作用的实验研究

Primary sj?gen's Syndrome (pSS) is s a chronic autoimmune disease characterized by infiltration of lymphocytes into exocrine gland, such as lacrimal and salivary glands, and clinically have symptoms of dry eyes and dry mouth.Currently,the main effect, glucocorticoids and immunosuppressive therapy, is not yet ideal. Interleukin -10 (IL-10) is a cytokines which was originally found to be produced by type 2 helper T cell (Th2), and inhibits the mRNA transcription of type 1 helper T cell (Th1). In recent years, it has been found that IL-10 has the function of stimulation and suppression to various cells. IL-10 is not only able to induce peripheral tolerance, but also has an important immunomodulatory function that inhibits neutrophil and eosinophil. In addition,it also inhibits the expression of monocyte MHC II molecules and antigen presenting role.So it is an important anti-inflammatory medium and immune suppression factors, and has closely relationship to inflammatory diseases, autoimmune diseases and transplantation diseases. Thus, there is a hot research about IL-10 for the autoimmune disease in recent years. Recent studies confirm that IL-10 has a therapeutic effect to the animal model of rheumatoid arthritis (RA). The adenovirus vectors can efficiently transfer exogenous genes into the target cells, while the "Sleeping Beauty" transposon can integrate exogenous gene into the genome. The present study intends to build the IL-10 gene "Sleeping Beauty" transposons combined with adenovirus vector system. The IL-10 gene is transferred to the mouse spleen cells, and the spleen cells can stably express IL-10.Then spleen cells with IL-10 were transfected in NOD mice subcutaneous, and the expression of IL-10 and other cytokines were detected in NOD mice. Our study will provides a new idea for treatment of pSS.

原发性干燥综合征(pSS)是一种以外分泌腺受累为主要特征的自身免疫性疾病。目前，临床上以糖皮质激素和免疫抑制剂治疗为主，效果尚不理想。白介素-10（IL-10）是一种重要的抗炎介质和免疫抑制因子，与炎症疾病、自身免疫性疾病等多种疾病密切相关。 腺病毒载体能高效的将外源基因直接转移到靶细胞内，而"睡美人"转座子能将外源基因整合到靶基因组中。本研究拟构建针对IL-10基因的"睡美人"转座子与腺病毒结合的载体系统，将IL-10基因转入到小鼠脾细胞中，使其能持续表达IL-10。将转染后的脾细胞种植到NOD小鼠皮下，检测处理过的NOD小鼠体内IL-10等相关细胞因子的表达，从而探讨IL-10在pSS发病机制中的作用及含有IL-10基因转座子/腺病毒载体系统在pSS动物模型中的治疗作用，为pSS的治疗提供新的思路。

干燥综合征（Sjögren's syndrome，SS）是一种以淋巴细胞浸润为特征的自身免疫性疾病，由于外分泌腺分泌减少，临床上主要表现为口、眼干燥等症状。非肥胖糖尿病（non-obese diabetes，NOD）小鼠可自发地出现外分泌腺淋巴细胞的浸润，血清中可检测到多种自身抗体和炎性细胞因子，因此，NOD小鼠是目前公认的SS的动物模型。本研究将腺病毒介导的“睡美人（SB）”转座子与IL-10共表达感染小鼠脾细胞。感染48h后将脾细胞种植到NOD小鼠右后腿的腘窝皮下。注射结束后4周处死小鼠，ELISA法检测各组NOD小鼠血清中IL-10、IFN-γ等细胞因子的表达水平；流式细胞术检测各组NOD小鼠脾细胞中CD4+IL-10+、CD4+IFN-γ+、Th17和Treg细胞的比例；HE染色检测小鼠唾液腺和泪腺组织的病理改变；荧光免疫组化检测小鼠唾液腺和泪腺组织中CD4、CD8、Foxp3的变化。研究发现：与对照组和空载体组比较，治疗组C57BL6小鼠脾细胞的IL-10 mRNA的表达水平明显升高，NOD小鼠血清中IL-10和IFN-γ的表达水平明显升高，NOD小鼠脾细胞中CD4+IL-10+细胞和Treg细胞的比例明显升高；而治疗组小鼠脾细胞中Th17细胞比例明显下降；治疗组NOD小鼠唾液腺和泪腺组织中淋巴细胞的浸润明显减轻；治疗组中NOD小鼠唾液腺和泪腺中CD4、CD8的比例下降，Foxp3的比例没有明显差异。综上，腺病毒介导的SB转座子与IL-10共表达后可以升高NOD小鼠血清中IL-10的表达水平；同时可明显地升高NOD小鼠脾细胞中CD4+IL-10+细胞的比例、降低Th17细胞的比例、升高Treg细胞的比例，减轻NOD小鼠唾液腺和泪腺中淋巴细胞的浸润；降低NOD小鼠唾液腺和泪腺组织中CD4、CD8的比例，调控Th1/Th2和Th17/Treg的平衡，对NOD小鼠起到治疗作用。因此，腺病毒介导的SB转座子与IL-10共表达可以调控NOD小鼠脾细胞Th1/Th2和Th17/Treg细胞平衡、减轻血清中细胞因子和外分泌腺炎症反应的影响，本研究也阐明IL-10对NOD小鼠的可能治疗机制，为SS的治疗提供了实验依据。