CD40L诱导严重烧伤后肺微血管内皮细胞损伤的机理研究

High mortality rate and thorny treatment of post-burn patients with acute lung injury (ALI) challenged burn clinic, thus explore of underling mechanisms of post-burn ALI and protection approach is important. Pulmonary microvascular endothelial cells (PMVECs) play a critical role in pathophysiology of ALI after severe burns, so researchers pay more and more attention to how to protect PMVECs in ALI. Abnormally high circulating level of CD40L was found in severe trauma, but the inherent relationship between CD40L and ALI is still unclear. Our previous studies showed that GEF-H1/RhoA signaling pathway activation is an important factor in production of inflammatory cytokines and hyperpermeability of vascular endothelial cells. In the present study, CD40L not only could significantly upregulate GEF-H1 and cleaved caspase-3 expression in PMVECs, but also could significantly reduce the expression of Syndecan-1 and VE-cadherin on the surface of PMVECs. The present study try to improve the hypothesis that CD40L-CD40 signal activates GEF-H1/RhoA signaling pathway, causing PMVECs barrier dysfunction and inflammatory and apoptotic cascades. The research results are benefit to understand mechanisms of the damage of PMVECs in post-burn ALI, and to explore potential strategies to prohibit CD40L-related post-burn ALI.

严重烧伤所致的急性肺损伤（ALI）治疗棘手且死亡率高，因而以肺微血管内皮细胞（PMVECs）为靶目标的防治研究具有重要的临床应用价值。严重创伤后循环中CD40L水平异常增高，但CD40L与伤后ALI是否存在内在关系尚不清楚。我们前期研究发现GEF-H1/RhoA信号通路的活化是血管内皮细胞炎症因子表达以及通透性增加的重要因素之一，而CD40L不仅能上调PMVECs内GEF-H1的表达和cleaved caspase-3水平，而且明显减少细胞表面Syndecan-1和VE-cadherin的表达。因此本研究推测CD40L与CD40结合后，活化胞内GEF-H1/RhoA信号通路，造成PMVECs屏障完整性和功能损伤，激活炎症和凋亡反应，从而探讨CD40L诱导烧伤后ALI的病理生理机制，并调控CD40L功能，以期改善PMVECs损伤，为严重烧伤后ALI的防治提供一种新的、潜在的临床治疗手段。

1..明确CD40L在体内肺组织损伤中的作用和重要地位；.2..探讨GEF-H1/RhoA信号通路在CD40L介导肺损伤中的作用，并将GEF-H1/RhoA信号通路作为干预靶点，以期改善CD40L诱导的肺组织损伤的研究；.【方法】.1. 在成年雄性C57/BL小鼠体内通过HE染色、Evans blue法、称重等方法，观察肺 部炎症情况及肺血管通透性的变化，以明确CD40L在小鼠体内诱导肺损伤中的作用和重要地位；.2. ①培养正常HPMVECs，通过蛋白电泳GEF-H1/RhoA/ROCK蛋白表达；蛋白芯片检测细胞内信号通路活化情况；定量PCR检测多种炎症因子的释放、G-LISA法检测RhoA活性；免疫荧光染色法检测细胞骨架结构及多种细胞连接蛋白的定位及表达；Transwell法检测CD40L对单层融合内皮通透性的影响；②利用拮抗剂、RNA干扰技术或特异性抑制剂分别拮抗或下调GEF-H1、RhoA、ROCK以及MAPK(p38、JNK)的表达或者活性，检测PMVECs通透性、凋亡反应、炎症因子表达的变化；.3. 使用CRE::Cas9双阳性小鼠，构建NC/GEF-H1/RhoA sgRNA及联合使用肺高度亲和的包装质粒，包装AAV病毒，通过尾静脉注射特异性敲除肺微血管内皮中GEF-H1/RhoA表达；②在NC sgRNA组、GEF-H1 sgRNA组、RhoA sgRNA组中，分别通过气管注射的方式给予鼠源CD40L的刺激，分别通过HE染色观察肺部炎症细胞浸润、Evans Blue法检测肺血管通透性变化，评估将GEF-H1/RhoA信号通路作为干预靶点以改善CD40L诱导肺微血管内皮损伤的作用；.【结果】.1..CD40L可诱导小鼠急性肺损伤发生；.2..CD40L可诱导HPMVECs中多条信号通路活化，炎症因子表达增加，内皮通透性增加及细胞内凋亡蛋白表达上调；.3..CD40L可活化GEF-H1/RhoA/ROCK信号通路,且通过此通路调控2中的效应；.4..抑制GEF-H1/RhoA/ROCK信号通路可以减轻CD40L诱导的小鼠急性肺损伤；.【结论】CD40L通过活化GEF-H1/RhoA/ROCK信号通路，介导内皮炎症效应、细胞骨架变化及细胞连接结构破坏，并促进细胞凋亡效应，从而介导cd40l诱导的急性肺损伤效应。