整合素连接激酶在新生鼠缺氧缺血脑损伤血管修复的信号调控

Hypoxic-ischemic encephalopathy (HIE) is one of the common and severe neonatal diseases. Angiogenesis has been shown to play an important role in the repairment of brain tissues after hypoxic-ischemic brain injury (HIBD). Recently, integrin-linked kinase (ILK) has been reported to be involved in the regulation of cellular processes including cellular repairment after injury, migration and angiogenesis via protein kinase B (PKB) pathway. However, it is not known whether ILK is involved in cerebral vascular reconstruction and the possible mechanisms in the neonatal rat brain after hypoxia-ischemia. Is there any internal relationship between ILK/PKB signaling pathway mediated-angiogenesis and vascular endothelial growth factor (VEGF)? Is Girdin, a protein mediated-cellular migrarion and PKB substrate, relevant to ILK/PKB signaling transduction? What is the role of Girdin in the signaling pathway? What are the relashionship among ILK/PKB, Girdin and VEGF in angiogenesis after HIBD? All of these scientific questions need to be targeted. In this project, we will establish an HIBD neonatal rats model and an in vitro hypoxic-ischemic model with cultured vascular endothelial cells to investigate the expression of ILK. With these models, we will explore the signaling transduction mechanisms of ILK/PKB, Girdin and VEGF in cell proliferation, migration and vascular reconstruction using knockout or over-expression of ILK or Girdin or PKB. We belive that our studies will provide new insights in the therapeutic strategies of neonates with HIE.

缺氧缺血脑损伤(HIBD)后，血管再生是脑组织自身修复的重要环节。近年研究发现整合素连接激酶(ILK)可通过蛋白激酶B(PKB)信号通路参与细胞损伤后的修复、迁移以及血管再生等过程。但新生鼠脑缺氧缺血后，ILK是否参与脑血管的修复重建？其可能的机制是什么？ILK/PKB信号通路介导的血管再生与血管内皮细胞生长因子(VEGF)间有无内在联系？新近发现的可介导细胞迁移的PKB底物Girdin蛋白在ILK信号传递中有无关联？起什么作用？二者与VEGF介导的血管再生间的关系均不清楚。本课题将通过建立新生鼠HIBD模型及体外脑血管内皮细胞缺氧缺血模型，观察ILK在血管内皮细胞中的表达，应用ILK、Girdin敲除或高表达方法及PKB信号通路抑制剂，研究ILK/PKB，Girdin及VEGF在介导血管内皮细胞增殖、迁移和血管修复重建中的信号调控机制，为临床治疗HIE提供新思路。

建立了新生大鼠缺氧缺血模型及体外培养大鼠肾上腺嗜铬细胞瘤（PC12）细胞，模拟神经元氧糖剥夺（OGD）模型，阐明了不同程度缺氧缺血情况下，信号转导和转录激活因3（STAT3）、磷酸化 STAT3、血管内皮生长因子（VEGF）、c-Jun氨基末端激酶（JNK)、FOXO3a、磷酸化FOXO3a、长链非编码 RNA BC088414、肾上腺素受体 β2（Adrb2）和凋亡蛋白Bim、CC3、半胱氨酸蛋白酶 6（Casp6）的表达及其与神经元凋亡的关系。进一步应用JNK 特异性抑制剂AS601245，小分子干扰 RNA（siRNA）抑制BC088414 表达；采用 TUNEL 法检测细胞凋亡情况，观察其对下游凋亡蛋白及Adrb2的调控，明确了JNK/FOXO3a/Bim通路及BC088414 通过调节凋亡蛋白Bim、CC3、Casp6及Adrb2的表达参与缺氧缺血后神经元凋亡过程。通过以上研究，深入阐明了不同通路（STAT3/p-STAT3/VEGF, JNK/FOXO3a/Bim及BC088414/Adrb2/Casp6）及其下游信号分子与神经元凋亡的关系，为逆转缺氧缺血性脑损伤提供了新靶点，为临床治疗新生儿缺氧缺血性脑病提供了新思路。发表中英文论文6篇（其中SCI论文4篇）。获得2014年四川省医学青年科技奖一等奖1项（新生儿缺氧缺血性脑病的修复机制）。