慢性移植排斥反应诱导的小鼠肾炎发生因素的初步探讨

Determination of pathological factors involved in lupus nephritic mice with cGVHD

目的探究慢性移植排斥反应诱导小鼠肾炎的发生机制,为下一步药物治疗提供实验依据。方法建立DBA/2→B6D2F1(DBA/2J×C57BL/6)移植小鼠模型,通过尿蛋白检测以及组织病理学分析确定小鼠肾炎的发生;利用直接免疫荧光标记技术以及实时定量PCR分析慢性移植排斥反应诱导小鼠肾炎发生过程中T、B淋巴细胞的活化情况,以及靶组织器官中各种因子或蛋白的变化。结果与同系基因型移植对照组(B6D2F1→B6D2F1)小鼠相比,异体移植实验组(DBA/2→B6D2F1)小鼠脾细胞中CD4+CD69+、CD8+CD69+双阳性细胞百分比均明显增加,B220+I-Ab+双阳性细胞的I-Ab表达显著增强,提示T、B淋巴细胞的活化;另外,在实验组小鼠肾组织中,趋化因子如单核细胞趋化蛋白-1(MCP-1)、受激活调节正常T细胞表达和分泌因子(RANTES)、B淋巴细胞趋化因子(BLC)以及淋巴细胞趋化因子(Ltn)表达上升;炎性细胞因子(如IL-6、IL-1β)表达显著增强;促纤维生长因子如转化生长因子β1(TGF-β1)、结缔组织生长因子(CTGF)以及纤维增生相关蛋白如Ⅰ型胶原蛋白(collagenⅠ)的mRNA表达水平明显上升,提示组织炎症以及纤维化的发生。结论慢性移植排斥反应诱导肾炎发生可能与激活淋巴细胞以及炎症因子、趋化因子和促纤维生长因子的分泌表达有关。

Objective To determine several pathogenic factors involved in the development of lupus nephritis in mice induced by chronic graft-versus-host disease（cGVHD）.Methods Expression of antigen CD69 in CD4＋ or CD8＋ T cells and expression of I-Ab in CD45R/B220＋ B cells of mouse splenocytes were tested using flow cytometric analyses.Changes in the mRNA level of several chemokines,cytokines,and protein in mouse kidneys were examined by real-time quantitative PCR technique.Results The percentage of both CD4＋CD69＋ and CD8＋CD69＋ double positive cells in the splenocytes of mice undergoing lupus nephritis induced by cGVHD was increased significantly compared with control group.The expression of I-Ab in CD45R/B220＋ B cells was also enhanced as shown by the observation that the mean fluorescence of I-Ab-PE staining in CD45R/B220＋ B cells in experimental group mice nearly quadrupled compared with control group.The transcript levels of several chemokines,cytokines and protein,including monocyte chemoattractant protein-1（MCP-1）,regulated upon activation,normal T-cell expressed and secreted（RANTES）,B lymphocyte chemoattractant（BLC）,lymphotactin（Ltn）,IL-6,IL-1β,transforming growth factor-beta 1（TGF-β1）,connective tissue growth factor（CTGF） and collagen Ⅰ were demonstrated to be up-regulated in nephritic mice.Conclusion All these results indicate that both T and B cells are activated in nephritic mice induced by cGVHD.Many proinflammatory factors,chemoattractants,as well as fiber-forming factors are involved in the pathogenesis of lupus nephritis in experimental group mice.