IL-35调控Th17/Treg细胞分化在改善胰岛移植疗效中的应用

In our previous study, we used RNA interference technique to silence inducible nitric oxide synthase (iNOS) gene in cultured pancreatic islets and found these islets had resistance to the impairment of cytokines IL-1β、TNF-α and some pro-inflammatory cytokines, which may ameliorate the viability and function of islets and beneficial to pancreatic islet transplantation. Along with the deepening and permeating research, we realized that T help cell 17 (Th17) and the regulatory T cells (Tregs), or the relevant active cytokines had a close and extensive correlation with the pancreatic islet graft function exhastion in early stage after transplantation. And the imbalance of Th17/Tregs differentiation maybe an important factor in the pathogenesis and development of typeⅠdiabetes mellitus. IL-35 is an latest discovered important cytokine which can regulate the differentiation of Th17/Treg cells. This subject is based on preceding model of pancredtic islets culture in vitro. The isolated pancreatic islet cells and the spleniclymphocyte from mice of different breeds were cocultured, and then a mouse model in vivo of pancreatic islet transplantation through liver portal vein was established. Regulating the relevant active cytokines such as IL-35、TGF-β、IL-6 etc, or using RNA interference tecnique to inhibite the Th17 critical transcription factor RORγt, or transfecting the IL-35 gene into receptor mouse by hydrodynamic-based gene delivery and lentiviral vector, we attempt to promote the proliferation of Treg cells, diminish the Th17 cells, and induce the differentiation from Th17 cells to Treg cells. Consequently, we hope to inhibit the rejection and the non-specific inflammatory reaction in pancreatic islet transplantation by regulating TH17 differentiation and inducing the Th17 cells to transform to the Treg cells. Discussing the application value of the regulation in the improvement of the viability and function of islets graft, ameliorating pancreatic islet transplantation, and trying to find an effective method to increase the result of treatment of diabetes mellitus by pancreatic islet transplantation.

在前期研究中，我们通过RNA干扰实验发现IL-1β、TNF-α等细胞因子可通过诱导iNOS过度表达，严重损害胰岛移植物的存活和功能。已有研究表明，Th17和Treg细胞及其相关细胞因子与胰岛移植的早期功能衰竭，及1型糖尿病的发生发展，均有密切而广泛的联系。IL-35是最新发现的调控Th17/Treg细胞分化的重要细胞因子。本课题以前期的胰岛体外培养模型为基础，将不同种系小鼠胰岛与淋巴细胞混合培养，并建立小鼠经门静脉胰岛移植模型。通过改变IL-35、TGF-β等细胞因子环境，RNA干转录因子RORγt，或使用流体力学注射和慢病毒载体将IL-35基因转染受体等手段，调控Th17/Treg细胞分化，促进Treg增殖，抑制Th17产生并诱导其向Treg转化。探讨这种调控在改善胰岛移植效果中的应用价值，力图找到能够抑制胰岛移植中的排斥反应和非特异性炎症反应的有效办法，改善胰岛移植治疗糖尿病的效果。

前期的研究中，我们通过RNA干扰实验发现IL-1beta、TNF-a等细胞因子可通过诱导iNOS过度表达，严重损坏胰岛移植物的存活和功能。已有研究表明，Th17和Treg细胞及其相关细胞因子与胰岛移植的早期功能衰竭，及1型糖尿病的发生发展均有密切而广泛的联系。IL-35是新近发现的调控Th17/Treg细胞分化的重要细胞因子。本课题以前期的胰岛体外培养模型为基础，将不同种系小鼠胰岛与淋巴细胞混合培养，并建立小鼠经肾被膜下胰岛移植模型。通过改变IL-35表达，AAV介导RNA干扰技术沉默转录因子RORyt等手段，调控Th17/Treg细胞分化，促进Treg增殖，抑制Th17产生。探讨这种调控在改善移植效果中的价值，力图找到抑制胰岛移植排斥反应和非特异性炎症反应的有效办法。体外实验结果显示，共培养系统中的Th17细胞计数增加，在施用IL-35后，与对照组相比，Treg细胞的数量显着增加（第五天IL-35组总CD4 + T细胞50.7％,对照组9.5％），而 Th17细胞的增殖率显著受到抑制（第五天IL-35组0.3％，对照组7.2％）。 而降低Th17 / Treg比率则可以显着改善了移植胰岛的功能。体外结果显示术后每天腹腔注射IL-35（10ug/day）可延长胰岛移植物存活时间（对照组8天，IL-35组25天），而利用rAVV9-RORγt-siRNA表达载体沉默受体小鼠脾脏淋巴细胞RORγt基因后，胰岛移植物存活时间明显延长（GFP组14天，rAVV9-RORγt-siRNA组35天）。脾脏淋巴细胞流式结果显示，沉默脾脏淋巴细胞RORγt基因后Th17细胞分化明显减少，而Treg细胞则增多（Th17细胞：RORγt组1.23％，GFP组6.82％，NS组7.76％，对照组2.92％；Treg细胞：RORγt组8.17％，GFP组2.90％，NS组2.83％，对照组4.36％）。体内糖耐量实验和免疫组化结果显示，Treg细胞增多可改善移植胰岛功能。综上结果表明，IL-35可通过调控Th17/Treg比例，可减轻胰岛移植术后排斥反应，改善移植胰岛功能。rAVV9-RORγt-siRNA可通过抑制CD4+T细胞向Th17细胞分化，增加Treg细胞数量，改善胰岛移植疗效。