微管相关蛋白4（MAP4）对血管通透性的调节作用及其机制研究

Abnormal vascular permeability plays an important role in the physiopathologic process of shock and sepsis after burn injury, and restoring the normal function of blood vessel will be critical for prevention and cure of these diseases..In the committed NSFC in operation, we have proved the MAP4 phosphorylation induced by hypoxia results in microtubules depolymerization in cardiomyocytes, and the MAP4 phosphorylation also increased after hypoxia or LPS treatment in HUVECs accompanied by elevated permeability. Considering the pivotal role of MAP4 phosphorylation in the cytoskeleton regulation, and microtubule and actin cytoskeleton are involved in the contractility of cells, we hypothesise that MAP4 is a key permeability regulation protein, and the p38/MAPK activation and followed MAP4 phosphorylation induced by hypoxia or LPS lead to microtubule and actin rearrangement and increase of vessel permeability. .In this study, we will firstly define the phosphorylated sites of MAP4 induced by hypoxia or LPS in HUVECs, then make clear the effect of MAP4 phosphorylation on the cytoskeleton and vessel permeability through constructively activating/inactivating MAP4 at the defined sites; Secondly, investigate the key pathway which induces MAP4 phosphorylation when shock or sepsis; thirdly, clarify the interactions between microtubule and actin when regulated by MAP4 simultaneously; at last, we will confirm the effect of MAP4 on vessel permeability on constructive activated MAP4 transgenic mice..This work aims to provide novel insights into the pathogenic mechanisms of abnormal vascular permeability during shock or sepsis, and offer feasible targets for clinic treatment for related diseases.

我们在青年基金中证实MAP4磷酸化是缺氧导致微管解聚的关键环节,据此发表论文一篇（CMLS, IF：7.05）。.血管通透性升高在休克/脓毒症中普遍存在，有效降低通透性是防治此类疾病的关键。本项目基于内毒素或烧伤血清处理的内皮细胞中MAP4磷酸化上调并伴随血管通透性增加的研究基础，结合细胞骨架重组是通透性改变结构基础的报道，提出“MAP4是一种血管通透性调节蛋白；烧伤休克或脓毒症期，缺氧或内毒素等刺激因素激活内皮细胞中p38激酶，使MAP4磷酸化而失活，导致微管微丝重分布和血管通透性增加”的科学假设。.为求验证，拟观察休克/脓毒症时MAP4磷酸化位点；构建该位点激活/失活细胞模型，证实磷酸化对细胞骨架和通透性的调节作用；明确导致其磷酸化的激酶通路；阐明MAP4调节微丝/微管的交互作用；在转基因小鼠上证明其对血管通透性的作用，以期为血管通透性调控提供新的理论基础，并为临床干预提供新思路。

肺微血管内皮细胞屏障破坏导致肺微血管通透性增高，进而引起急性肺损伤。肺微血管通透性增高机制尚不清楚。微管在内皮细胞通透性调节中具有重要作用，微管相关蛋白4 (MAP4)通过磷酸化的方式来调节自身活性，磷酸化增高时，MAP4活性下降，从微管上脱落导致微管失稳。本研究旨在明确MAP4磷酸化在炎症引起的HPMECs通透性增高中的作用及其相关机制，以期为临床相关疾病的治疗提供新的靶点和研究方向。.致炎因子LPS (200、500、1000ng/ml) 或 TNF-α (200、500、1000ng/ml) 处理HPMECs 6h后，p38/MAPK激活，MAP4(S696与S787)磷酸化增高, 而MAP4 (Ser768)磷酸化无改变；同时单层HPMECs荧光物质漏出增多，跨内皮细胞电阻值降低，微管断裂，游离态微管蛋白增多并且呈剂量依赖性改变。微管稳定剂紫杉醇 (1uM)预处理或MAP4 (Ala)转染可以减少致炎因子导致的荧光物质漏出，跨内皮细胞电阻值明显增加，微管网状结构破坏减少，聚合态微管蛋白含量增加。转染MKK6 (Glu)持续激活p38/MAPK后，MAP4 (S696与S787)磷酸化水平显著增加，MAP4与微管相互结合减少，单层HPMECs荧光物质漏出增多，跨内皮细胞电阻值降低，微管解聚增加。p38/MAPK阻断剂SB203580 (5uM)预处理细胞后，致炎因子刺激导致的上述变化得到明显抑制。caspase-3抑制剂（Z-DQMD-FMK, 10uM）预处理细胞减少致炎因子诱导的细胞凋亡；但SB203580 (5uM)预处理或MAP4 (Ala)转染可使致炎因子刺激所致的HPMECs荧光物质漏出继续减少，跨内皮细胞电阻值仍有增加，提示MAP4磷酸化介导的致炎因子所致HPMECs通透性增加不依赖细胞凋亡。.综上所述，致炎因子处理HPMECs后，激活 p38/MAPK通路，MAP4 (S696与S787)磷酸化而失活，引起微管解聚，导致HPMECs通透性增高；MAP4磷酸化介导的HPMECs通透性增高不依赖细胞凋亡。以上结果有助于我们进一步深入认识炎症时急性肺损伤的发病机理，为临床相关疾病的干预和治疗提供新靶点和方向。