Wnt信号转导通路在高氧CLD早产鼠肺泡上皮细胞损伤后修复异常中作用机制的研究

To study Wnt signal transduction in chronic lung disease induced by hyperoxia in premature rats and its regulation mechanism of proliferation of typeⅡ alveolar (epithelial) cells（AEC-Ⅱ）and its differentiation to type I alveolar (epithelial) cells (AEC-I ). We adopt the ready-made CLD models induced by hyperoxia in premature rats. Meanwhile, we culture original AEC-Ⅱof these models and AEC-I differentiated from AEC-Ⅱ.Using PCR,Western-Blotting,transfection in cell, co-immunoprecipitation and other molecular biology or cytobiology technologies to study:1) Different kind of Wnts switch on three（Wnt/β-catenin、Wnt/PCP、Wnt/Ca2+） different signal transductions, to explore the expressing patterns of their main genes and proteins in lung tissue of normal and CLD premature rats and AEC-Ⅱ. 2)Changes of AEC-Ⅱ ability to proliferation and differentiation to AEC-Ⅰby changing activity of Wnts switching on different signal transductions. Trying to confirm: Abnormality of Wnt signal transductions in activition, inhibition or disturbance of their balance would result in disorder of generation cycle of AEC-Ⅱand decreased ability to differentiation to AEC-Ⅰ, which was one of the most important mechanisms of alveolar epithelial cell dysreepithlialization in chronic lung disease induced by hyperoxia.It should be theoretical and experimental foundation to prevent and treat CLD in newborns in early time.

为研究高氧致慢性肺疾病（CLD）早产鼠Wnt信号通路对Ⅱ型肺泡上皮细胞（AEC-Ⅱ）增殖及其向Ⅰ型肺泡上皮细胞（AEC-Ⅰ）转化的调控机制。采用已建立的高氧诱导早产鼠CLD模型及体外培养AEC-Ⅱ及其转化的AEC-Ⅰ，应用PCR、蛋白印迹、细胞内转染、免疫共沉淀等多种分子、细胞生物学技术，研究：1）不同种类Wnt启动的（Wnt/β-catenin、Wnt/PCP、Wnt/Ca2+）三条通路中主要基因和蛋白在早产鼠正常肺发育及CLD发生中的肺组织以及AEC-Ⅱ中表达规律；2）改变Wnt启动的三条途径活性对CLD早产鼠AEC-Ⅱ增殖转化的影响。试图证实：Wnt信号通路异常激活、抑制、它们之间平衡关系破坏而导致AEC-Ⅱ的细胞周期调控紊乱以及AEC-Ⅱ分化为AEC-Ⅰ能力降低是高氧CLD肺泡上皮细胞损伤后修复障碍和肺泡化障碍的重要机制之一。为早期有效防治早产儿CLD奠定理论与实验基础。

为研究高氧致慢性肺疾病（CLD）早产鼠Wnt信号通路对Ⅱ型肺泡上皮细胞（AEC-Ⅱ）增殖及其向Ⅰ型肺泡上皮细胞（AEC-Ⅰ）转化的调控机制。采用已建立的高氧诱导早产鼠CLD模型及体外培养AEC-Ⅱ及其转化的AEC-Ⅰ，研究：① 不同种类Wnt启动的（经典/非经典）通路中主要基因和蛋白在早产鼠正常肺发育及CLD发生中的肺组织以及AEC-Ⅱ中表达规律；② 改变Wnt活性对CLD早产鼠AEC-Ⅱ增殖转化的影响 。重要结果: 高氧CLD肺泡上皮细胞损伤后修复障碍中，非经典通路Wnt5a/PCP受抑制，经典通路Wnt3a/β-catenin异常激活，其与Wnt5a之间的平衡关系被破坏；具体表现为：① 一方面Wnt3a 以及β-catenin表达明显增多；AEC-Ⅱ 在高氧暴露后，Wnt3a/β-catenin信号通路被激活，AEC-Ⅱ增殖明显而转化障碍；细胞水平敲除Wnt3a可以通过β-catenin/TCF阻断AEC-Ⅱ向AEC-Ⅰ转化的抑制。② 另一方面，体内和体外研究均显示高氧暴露后AEC-Ⅱ 的Wnt5a表达明显下降与AEC-Ⅱ转化能力相关；腺病毒转染Wnt5a至高氧组AEC-Ⅱ，Wnt5a的过度表达和外源性增加其量可以抑制高氧对上皮细胞转化异常的影响。③ 外源性Wnt3a下调Wnt5a的表达，进一步致AEC-Ⅱ增殖明显而转化障碍。④ 白藜芦醇抑制Wnt/β-catenin 信号缓解高氧诱导的氧化应激，减轻炎症反应，缓慢纤维组织增生。.科学意义：Wnt信号通路异常激活、抑制、它们之间平衡关系破坏而导致AEC-Ⅱ的细胞周期调控紊乱以及AEC-Ⅱ分化为AEC-Ⅰ能力降低是高氧CLD肺泡上皮细胞损伤后修复障碍和肺泡化障碍的重要机制之一。为临床早期发现和探寻有效的CLD防治途径提供新思路。同时，干预因素白芦藜醇的应用效果为临床可能产生新的治疗药物奠定基础。