【作者】 王翠平；

【导师】 严金川；

【作者基本信息】 江苏大学 ， 临床检验诊断学， 2015， 博士

【摘要】 目的：(1)血管损伤所致的血管疾患目前是全球范围内发病率及死亡率最高的疾病之一。近来研究发现PI3Kγ在炎症、氧化应激、心血管疾病及组织损伤、修复中起重要作用。本研究旨在探讨PI3Kγ在小鼠血管损伤中的作用及机制。(2)越来越多的证据表明血小板参与血管炎症的发生发展,本研究旨在探讨血小板PI3Kγ在血管损伤中的作用及机制。方法：(1)在小鼠颈动脉结扎模型基础上比较野生型(WT)及PI3Kγ基因敲除小鼠血管损伤反应。采用病理形态学分析新生内膜形成情况,并用免疫荧光及免疫组化法检测损伤血管炎症细胞浸润及NF-κB表达,RT-PCR检测炎症介质的表达, DHE染色检测血管壁活性氧生成。体外实验中采用MMT法及细胞划痕实验、transwell小室迁移法比较野生型PI3Kγ基因敲除血管平滑肌平滑肌细胞增殖及迁移。(2)在小鼠颈动脉部分结扎模型上反复静脉注射活化的血小板。采用病理形态学分析颈动脉中膜-内膜厚度,免疫荧光法检测血管炎症细胞浸润,明胶酶谱法检测受损血管局部MMP-9的活性,实时荧光定量PCR检测损伤血管局部炎症介质包括炎症因子(TNF-α、IL-6)及粘附分子(VCAM-1,ICAM-1)的表达,DHE染色检测血管壁活性氧生成。体外实验中采用流式细胞术检测血小板表面分子表达、活性氧生成、血小板白细胞聚集物形成,western blot检测血小板Akt及p-38表达及活化,血小板、内皮细胞共培养检测血小板-白细胞相互作用。结果：(1)血管损伤后3天、7天白细胞浸润明显,其中第3天以中性粒细胞为主,第七天以单核巨噬细胞为主,而PI3Kγ基因敲除小鼠损伤血管局部炎症细胞浸润均明显减少；PI3Kγ基因敲除小鼠损伤血管局部的细胞因子(TNF-α和IL-6)及粘附分子(ICAM-1和VCAM-1)及NF-κB表达减少。(2)血管损伤后PI3Kγ基因敲除小鼠活性氧的生成较WT小鼠明显降低。(3)血管损伤后PI3Ky基因敲除小鼠新生内膜形成较WT小鼠明显减少,血管平滑肌细胞向内膜的增殖迁移减少；体外实验显示PI3Kγ-/-血管平滑肌增殖及迁移能力降低。(4)颈动脉部分结扎模型显示小鼠反复注射ADP活化的血小板后血管内膜-中膜增厚血管重塑明显,而注射PI3Ky-/-血小板后此作用明显减弱。(5)WT血小板注射小鼠损伤血管的炎症细胞浸润、炎症介质表达、MMP-9活性及活性氧生成明显增多；而PI3Kγ-/-血小板注射小鼠血管炎症细胞浸润、炎症介质表达、MMP-9活性及活性氧生成增多不明显。(6)体外实验研究发现ADP不能诱导PI3Ky-/-血小板Akt、p-38活化,不能诱导PI3Ky-/-血小板活化。(7)ADP不能诱导PI3Kγ-/-血小板NADPH酶活化及活性氧生成。(8)ADP诱导的血小板-白细胞聚集物形成及血小板-内皮细胞粘附中PI3Kγ-/-血小板较WT血小板明显减弱。结论：(1)PI3Kγ介导血管损伤后的炎症反应、活性氧生成及新生内膜的形成。(2)血小板PI3Kγ通过调节血小板活化、氧化应激及其与白细胞、内皮细胞的相互作用介导血管损伤后的炎症反应、活性氧生成及新生内膜形成。展望：PI3Ky可能作为心脑血管疾病的一个治疗靶点,同时血小板PI3Ky可作为心脑血管疾病中抗血小板治疗的一个新的研究方向。更多还原

【Abstract】 Objectives(1) Vascular disease is a significant cause of morbidity and mortality in developed countries and results from vascular injury. Increasing evidence reveals that phosphoinositide 3-kinase gamma (PI3Kγ) is a key regulator of inflammatory and oxidative responses and has been implicated in a number of inflammatory diseases, such as atherosclerosis and myocardial ischemia/reperfusion injury. In our study, we aim to investigate the contribution of PI3Kγ in vascular injury and repair.(2) Studies have begun to focus on the emerging function of platelets as immune and inflammatory cells that initiate and accelerate vascular inflammation. This study aims to investigate the role of platelet PI3Kγ in vascular remodeling under flow-disturbed conditions.Methods(1) Mouse left carotid artery ligation was used as vascular injury model. Pathological morphological analysis was performed. Leukocyte recruitment and NF-κB expression were assessed by immunofluorescence and immunohistochemistry staining. Proinflammatory mediator expressions were assessed by RT-PCR. Using the superoxide-sensitive dye dihydroethidium (DHE) staining technique to detected the production of reactive oxygen species. In vitro, the proliferation and migration of vascular smooth muscle cells (VSMCs) were assessed by MTT assay, wound healing and transwell migration assay.(2) Mouse partial left carotid artery ligation with adoptive transfer of activated, washed wild-type or PI3Kγ-/- platelets was used as the model to indetify the role of platelet PI3Kγ. Intima-media area was calculated. Leukocyte recruitment and proinflammatory mediator expression intercellular adhesion molecule-1(ICAM-1), vascular cell adhesion molecule-1(VCAM-1), tumor necrosis factor alpha(TNF-a), and interleukin-6 (IL-6) expression were assessed by immunofluorescence or RT-PCR. MMP-9 activity was assessed by gelatin zymography. Production of reactive oxygen species was detected by DHE staining. In vitro PI3Kγ-/- platelets were used to verify the effect of PI3Kγ on platelet activation, ROS production and interaction with leukocytes by flow cytometry. Platelets were co-cultured with endothelials to verify the interation between platelets and endothelials.Results(1) In wild-type mice, neutrophil infiltration was dramatically increased at 3d, while macrophage infiltration was dramatically increased at 7d after carotid injury. In PI3Kγ-/- mice, absence of PI3Ky profoundly reduced the recruitments of neutriphil and macrophage. Comparied with WT mice, the expression of NF-kB and the mRNA expression of cytokines (TNF-a and IL-6), adhesion molecules (ICAM-1 and VCAM-1) in vascular wall were markedly blocked after vascular injury.(2) ROS production in the ligated artery wall was reduced in PI3Kγ-/- mice comparied with WT mice.(3) PI3Ky deficiency reduced the neointima formation and VSMC proliferation and migration to intima. In vitro, PI3Kβ deficiency inhibited VSMC proliferation and migration.(4) Mice injected with activated platelets showed a significant increase in intima-media thickening, these effects were abrogated by platelet PI3Kγ deficiency.(5) Mice injected with PI3Kγ-/-platelets showed reduced recruitments of neutrophils (at 3 d) and macrophages (at 21 d); lower expressions of ICAM-1, VCAM-1, TNF-a, and IL-6 expression (at 3 d); and lower MMP-9 activiy and ROS production in the flow-disturbed area comparied with WT platelets injected mice.(6) In vitro, PI3Kγ deficiency inhibited Adenine di-Phosphate -induced platelet activation through the Akt and p38 MAP kinase signaling pathways.(7) PI3Kγ deficiency inhibited ADP induced NADPH activation and ROS production in vitro.(8) Platelet PI3Kγ deficiency reduced platelet-leukocyte aggregation in vivo and in vitro and platelet-endothelial cell (EC) interaction in vitro.Conclusions(1) PI3Kγ plays a criticle role in neointima formation, vascular inflammation and ROS production response to vascular injury(2) Platelet PI3Kγ contributes to platelet-mediated vascular inflammation, ROS production and carotid intima-media thickening through mediate ADP induced platelet activation, ROS generation and interaction with leukocyte and endothelial cells.Prospect:PI3Kγ may be a new target for cardiovascular and cerebrovascular disease treatment. Anti-platelet PI3Kγ may be a new approach to anti-platelet in cardiovascular and cerebrovascular disease.更多还原