HMGB1下调HOXA9表达活化血管内皮细胞功能的分子机制

Activation of vascular endothelial cells leads to microcirculatory disturbance and thus causes multipie organ dysfunction which play an important role in the development of sepsis .High mobility group box protein 1 (HMGB1) is reported as a new and potential inflammatory mediator participated in sepsis. But the intracellularly signal transduction pathways and the detailed mechanism of how HMGB1 activates the vascular endothelial cells is still not clear. Transcription factors homeoboxA9( HOXA9 ) can be expressed inside the endothelial cells and their products act as a basis to maintain intracellular homeostatis. HOXA9 has been identi?ed as a key regulator of endothelia activation. In this study, we estabolished human umbilical vein endothelial cells （HUVEC） model to inhibit the expression of HMGB1 and observed how downregulation of HMGB1 affects the activity of HUVEC. By doing so, we hope to further demonstrate the importance of HMGB1 on activation of HUVEC. We also detected how the expression of HMGB1 activated by HUVEC affects the synthesis of HOXA9. We used EMSA, Chromatin Immunoprecipitation Assay, Tyrosine Kinase Inhibitor, etc to determine the signal transduction pathways of involving transcription factors and those which regulate the expression of HOXA9. Also we used RNA interference technique to evaluate the effects of co-diminution of both HMGB1 and HOXA9 to the activity of HUVEC. Eventually, by building up the animal models, we try to demonstrate the molecular mechanism of how HMGB1 downregulation affects the expression of vascular endothelial cells by HOXA9. We also hope to provide new clues to the treatment and prevention of sepsis combined with multiple organ dysfunction .

血管内皮细胞激活导致微循环障碍，是脓毒症发病机制的中心环节，高迁移率族蛋白B1（HMGB1）作为晚期炎性介质，参与了脓毒症的发病过程。但HMGB1激活血管内皮细胞的细胞内信号通路及机制目前仍所知甚少。转录因子同源盒蛋白A9（HOXA9）是维持血管内皮细胞稳定的基础，其表达下调是血管内皮细胞激活的必要条件。因此本实验拟建立抑制HMGB1表达的血管内皮细胞（HUVEC）模型，观察HMGB1表达降低对HUVEC活性的影响。检测HUVEC激活后HMGB1表达的变化对HOXA9表达的影响，通过凝胶电泳阻滞实验（EMSA），染色质免疫沉淀和激酶抑制剂等实验确定参与其中的转录因子和调节HOXA9表达的信号转导通路。并且利用RNA干扰技术，评价HMGB1和HOXA9共同消减后对HUVEC活性的影响。最后建立动物模型，阐明HMGB1下调HOXA9表达活化血管内皮细胞的分子机制，为脓毒症治疗提供新的思路。

血管内皮细胞激活导致微循环障碍，是脓毒症发病机制的中心环节，高迁移率蛋白B1(HMGB1)作为晚期炎症介质，参与了脓毒症的发病过程。但HMGB1激活血管内皮细胞的细胞内信号通路及机制目前仍所知甚少。转录因子同源盒蛋白A9(HOXA9)是维持血管内皮细胞稳定的基础。其表达下调是血管内皮细胞激活的必要条件。因此，本研究建立了抑制HMGB1表达的血管内皮细胞（HUVEC）模型，发现HMGB1表达降低的HUVEC的能明显抑制细胞的增殖和迁移。同时还成功构建了人的HMGB1基因的短发夹的真核细胞表达载体并且在人的脐静脉血管内皮细胞中有效的干扰了HMGB1的表达，短发夹的HMGB1基因真核细胞表达载体在人的脐静脉血管内皮细胞中促进了HOXA9的高表达和E-选择素的低表达。动物实验部分我们利用前期构建的干扰质粒和后期的小RNA质粒，减低了HMGB1的表达，分别打入动物体内，发现降低了HMGB1的表达，炎症因子和粘附分子的表达下降，组织的损伤程度降低，并且炎症指标粘附分子等表达下调且可能通过核因子KB。我们推论HMGB1和HOXA9可能是脓毒症治疗的新的靶点。