炎症早期局部内源性脂氧素生成调控SAP-SIRS的机制研究

Lipoxin A4 (LXA4) is called "inflammatory braking signal" for its remarkable anti-inflammatory effect. Our pilot studies also confirmed that LXA4 could improve prognosis of severe acute pancreatitis (SAP) by restraining local inflammation, reducing excessive Systemic Inflammatory Response Syndrome (SIRS), and ameliorating lung injury. However,LXA4 is mainly generated in the late stage of inflammation and the half-time is short . The quantity of LXA4 produced in the early stage is so little that it could hardly reverse the SIRS in SAP which developed promptly. Accordingly, we assume that to increase LXA4 synthesis in early stage of SAP by transfecting 15-lipoxygenase(15-LO), the key enzyme in LXA4 biosynthesis, to pancreatic acinar cells, could further inhibit the progress of inflammation and negatively regulate SIRS. Taking the advantage of genetic technology and transgenic animals, our subject aimed to overexpress 15-LO in the pancreas in the early stage of SAP. The effects of LXA4 on local inflammation and the mechanism of regulating SIRS activation and protecting the remote organ injury in SAP will be studied. It is the first study to regulate SIRS by promoting the expression of LXA4 in pancreas in the early stage of SAP, secondary to attend to control local inflammation, thus alleviating the remote organ injury. Our research not only provides novel ideas for the treatment of SAP, but also emphasizes the regulation of SIRS.

脂氧素A4（LXA4）因强烈的抗炎症作用被称为"炎症刹车剂"。我们前期研究证实给予外源性LXA4可抑制重症胰腺炎（SAP）局部炎症，减轻过度的全身炎症反应（SIRS），缓解肺损伤，改善预后。但LXA4半衰期短，多于炎症晚期产生，SAP炎症早期少量的LXA4对于此迅速发展为SIRS的疾病难起逆转作用。鉴此，我们设想"将LXA4合成的关键酶（15-LO）基因导入胰腺腺泡细胞，重组构建的启动子系统能使LXA4在炎症早期过度产生；从而抑制SAP炎症，负调控SIRS进展"。本课题借助基因转染技术和转基因动物，使15-LO在SAP启动早期表达，可研究LXA4对局部炎症及SIRS激活的调控，并评价此策略对胰外脏器的保护作用。我们首次尝试将LXA4合成的关键酶在炎症早期启动过表达，对局部炎症进行调控，并试图下调SIRS，改善脏器损伤。该课题不仅为SAP防治提供新思路，尤其突出了对SIRS的负调控。

体外实验： Alox15基因在脂氧素A4生成中的作用.目的：该实验阐明了LXA4生成的新途径。.方法：1.采用密度梯度离心法提取中性粒细胞。2.构建含Alox15基因的慢病毒。3.Alox15慢病毒转染血管内皮细胞。4.Alox15基因的血管内皮细胞与中性粒细胞在37摄氏度共培养5h之后,加入100nM浓度的FMLP刺激15min。ELISA法检测培养液上清液中脂氧素A4的浓度。.结果：1.成功提取外周血中的中性粒细胞。2.成功构建含Alox15基因的慢病毒，Alox15慢病毒成功转染血管内皮细胞。Real-time PCR法检测转染Alox15基因的血管内皮细胞15-LO mRNA稳定表达。3.转染Alox15基因的血管内皮细胞与中性粒细胞共培养后采用FMLP刺激可以产生LXA4。.结论 ：成功构建含Alox15基因的慢病毒载体,并实现该基因在血管内皮细胞内的稳定表达。含Alox15基因的血管内皮细胞与中性粒细胞共培养后，在FMLP的刺激下可以产生LXA4。.体内实验：Alox15基因对大鼠急性胰腺炎的治疗作用及其机制.目的：探讨Alox15基因对大鼠急性胰腺炎（acute pancreatitis，AP）的治疗作用及其可能的机制研究。.方法:雄性SD（Sprague-Dawley）大鼠24只，将其随机分成为4组，每组6只：正常对照组，急性胰腺炎组，Alox15慢病毒组，阴性对照病毒组。Alox15慢病毒组取5×107的Alox15慢病毒行胃左动脉灌注转染大鼠。大鼠胃左动脉灌注5天后，急性胰腺炎组、Alox15慢病毒组、阴性对照病毒组采用大剂量雨蛙素（caerulein）诱导建立大鼠急性胰腺炎模型。第一次雨蛙素腹腔注射后9小时抽血，留取血清、胰腺和肺标本，进一步的检查用。.结果：携带Alox15目的基因的慢病毒载体成功的转染大鼠。Alox15基因表达能够减轻急性胰腺炎的血淀粉酶浓度、血液中促炎细胞因子(IL-6 、MCP-1)浓度、胰腺和肺组织TNF-α、IL-1β、IL-6 、MCP-1的mRNA的水平、胰腺和肺湿干比、胰腺和肺病理学评分；减少胰腺和肺组织内中性粒细胞比例；增加胰腺腺泡细胞的凋亡；抑制胰腺和肺组织内NF-κB p65蛋白表达。.结论：转染Alox15基因对急性胰腺炎起治疗作用。