脓毒症急性肺损伤时HSPA12B在肺微血管内皮细胞中的表达及其作用机制

ALI/ARDS is one of most frequently complications of sepsis. There are no proven pharmacologic therapies to reduce the severity of lung injury or improve the clinical outcomes. Various studies show that protecting of pulmonary microvacular endothelial cells(PMVEC) can ameliorate ALI/ARDS. As HSPA12B which is predominantly expressed in endothelial cells is one member of heat shock proteins. It is reported that high expression of HSPA12B can attenuate cardiac dysfunction.Another study shows that HSPA12B promotes angiogenesis through suppression of AKAP12 and up-regulating VEGF pathway. Activation of VEGF pathway can enhance the permeability of vascular endothelial.We find that the expression of HSPA12B in cecal ligated and punctured(CLP) C57 mice lung tissue and LPS treated PMVEC is increased. We also find that the concentration of IL-1、IL-6 and TNF-a in the cell culture fluid of PMVEC is elavated when treated with HSPA12B siRNA and LPS. However, it haven't been fully elaborated why the expression of HSPA12B is increased in sepsis and what is the effect of HSPA12B to the PMVEC and how does the HSPA12B influence PMVEC. According to those questions, we intend to establish three different PMVEC which are HSPA12B high-expressed PMVEC, HSPA12B low-expressed PMVEC and HSPA12B normal-expressed PMVEC to evaluate the influence of HSPA12B to the function of PMVEC in vitro assessed by the expression of Ca-adhesin, E-selectin, ICAM-1 and the apoptosis of PMVEC and tranendothelial electrical resistance. We aslo use HSPA12B siRNA to interfere HSPA12B expression to appraisal the influence of HSPA12B to the function of PMVEC in vivo. We then investigate the possible mechanism of the effect of HSPA12B to PMVEC focusing on the kinases in different pathways including MLCK, Src, PKC and small GTPase using specific inhibitor both in vivo and in vitro.As the expression of HSPA12B in PMVEC treated by LPS is elavated and the TLR4 is the main receptor of LPS, We then study whether the activation of TLR4 signalling pathway has influences on the expression of HSPA12B in PMVEC.

维持PMVEC正常的结构和功能，可以降低脓毒症急性肺损伤严重程度。HSPA12B是一种主要表达在血管内皮细胞上的热休克蛋白。研究表明，HSPA12B高表达能减轻脓毒症时心功能障碍。我们的前期工作发现，脓毒症小鼠肺组织中HSPA12B表达上调；LPS处理的PMVEC中HSPA12B表达上调,HSPA12BsiRNA处理后，其培养液中炎症因子含量增加。但脓毒症时HSPA12B表达上调的机制及其对PMVEC功能的影响及机制，尚待研究。本课题拟通过TLR4受体缺陷的PMVEC研究TLR4受体及其下游信号通路，探索脓毒症时HSPA12B表达上调的机制；构建HSPA12B表达上调、下调和正常的PMVEC，LPS处理后检测相关信号通路激酶的表达及活性，并通过构建HSPA12B表达下调的小鼠,分别在离体和在体条件下探讨HSPA12B对PMVEC的影响及机制，以丰富对脓毒症时急性肺损伤发生发展的认识。

HSPA12B是一种主要表达在血管内皮细胞上的热休克蛋白，是一种急性期反应蛋白。肺微血管内皮细胞（PMVEC）是脓毒症急性肺损伤的关键细胞之一。如果能维持PMVEC正常的结构和功能，则可能降低脓毒症急性肺损伤严重程度。本课题探讨脓毒症急性肺损伤时HSPA12B在肺微血管内皮细胞中的表达及其作用机制。一、构建在体盲肠穿孔结扎（CLP）鼠模型和LPS刺激鼠肺微血管内皮细胞的离体实验，检测HSPA12B在各时间点的表达水平；用小干扰RNAs（siRNAs）下调mPMVECs中HSPA12B表达后，观察mPMVECs迁移和凋亡的变化，以及检测LPS刺激mPMVECs产生的炎症因子，并探讨其与MAPK信号通路中p38的关系。结果：（1）脓毒症时内毒素对鼠肺组织和mPMVECs中HSPA12B表达先升高后下降。（2）mPMVECs经LPS刺激24h后，HSPA12B表达下调组IL-6和TNF-α mRNA表达水平显著上升，而IL-10则表达下降。HSPA12B表达下调组的mPMVECs的迁移功能明显减弱、凋亡明显增多。说明HSPA12B可能对内毒素刺激的鼠肺微血管内皮细胞的具有一定的保护作用。（3）Western blot分析检测和免疫荧光双标和免疫共沉淀实验证实HSPA12B可能通过降低p38磷酸化水平来抑制LPS对内皮细胞的损伤。二、主要研究脓毒症时HSPA12B对血管内皮功能（通透性）的直接影响。结果表明：HSPA12B可能通过上调VE-cadherin的表达，降低血管内皮细胞的通透性，保护血管内皮的屏障功能。三、筛选和研究脓毒症肺损伤时对HSPA12B有调控作用的miRNA。通过生物学信息技术和miRNA mimics转染以及用LPS刺激，确定hsa-miR-4505是脓毒症时对人HSPA12B基因有表达调控作用的miRNA。研究结果表明NF-κB、JNK、ERK和P-38信号通路对hsa-miR-4505的表达具有调节作用，hsa-miRNA-4505可以通过降低HSPA12B的表达，使HUVEC屏障功能损伤加重。四、探讨HSPA12B对脓毒症肺血管内皮损伤小鼠生存率的影响及其可能的作用机制。经鼻吸入HSPA12B siRNA使脓毒症小鼠肺组织内HSPA12B表达下调，经过连续7天记录的生存率显示：HSPA12B干扰组小鼠生存率为0，明显低于NC siRNA组、CLP