胰脂肪酶家族在高甘油三脂血症性胰腺炎早期重症化中的作用及机制研究

The mechanism by which hypertriglyceridemia pancreatitis (HTGP) worsens is unknown. Studies showed that unsaturated fatty acid(UFA) generated via lipolysis of visceral adipocyte triglyceride（TG） by pancreatic lipases, caused the aggravation of AP in the obese mice.Pancreatic lipase, however, is a family of four lipases. The key enzymes in pancreatic lipase family members that mediate lipotoxicity and their role in HTGP worsening are still unknown. In order to solve this problem, this project intends to use two animal models with severe HTGP, GPIHBP1 knockout mice and high fat diet fed hamsters, which our previous studies found. We will knock down the expression of PTL, CEL, PLRP1 and PLRP2 in the pancreatic lipase family, respectively, by construct appropriate serotype of adeno-associated virus vector with high specificity to pancreatic acinar cells. Then the key enzymes causing severe HTGP will be defined on the basis of induction of HTGP. In addition, Orlistat was injected intraperitoneally as a control, to determine the effect and mechanism of key pancreatic lipase hydrolysis of chylomicrons in the pancreatic vessels and peripancreatic fat on the aggravation of HTGP. We studied the lipolysis of chylomicrons by pancreatic lipasesin vitro, and the effects of generated UFA and fatty acid chlorohydrins on the damage of acinar cells, and elucidated the mechanism of key lipase mediated cell damage. This study not only clarifies the key issues of severe HTGP, but also provides new treatment strategies for clinical prevention and treatment.

高甘油三脂血症性胰腺炎（HTGP）重症化的机制未明。有研究发现胰脂肪酶分解内脏脂肪细胞内的甘油三脂（TG）产生不饱和脂肪酸(UFA)导致肥胖小鼠AP重症化,然而胰脂肪酶家族包括4种酶，其在HTGP重症化中的关键酶和作用还不清楚。为了解决这个问题，本项目拟用我们前期构建HTGP重症化的两个动物模型，GPIHBP1基因敲除小鼠和高脂饲料喂饲的仓鼠，构建腺泡细胞特异性高的合适血清型的腺相关病毒载体，分别敲低胰脂肪酶家族中的PTL、CEL、PLRP1和PLRP2表达，在诱导HTGP的基础上，判断导致HTGP重症化的关键酶；然后以奥利司他腹腔注射为对照，探究关键酶分解胰血管乳糜微粒和胰周脂肪在HTGP重症化中的作用及机制。体外研究胰脂肪酶分解乳糜微粒产生UFA及脂肪酸氯醇损伤腺泡细胞的作用，并阐明关键脂肪酶介导细胞损伤的机制。本研究不但明确了HTGP重症化的关键问题，而且为临床防治提供新的治疗策略。

研究背景和目的：我们假说胰腺腺泡细胞释放的胰腺甘油三酯脂肪酶（Pancreatic triglyceride lipase ，PTL）水解乳糜微粒和脂肪细胞的甘油三酯（Triglyceride, TG）产生游离脂肪酸（Free fatty acids，FFAs）导致胰腺和胰外器官损伤是HTGP重症化的重要机制。. 研究方法：以糖基磷脂酰肌醇高密度脂蛋白结合蛋白1敲除（Gpihbp1-/-，简称GPI-/-）建立的HTG小鼠为对象，ICR野生小鼠（WT小鼠）为对照，采用腹腔注射雨蛙肽诱发急性胰腺炎(AP)，通过病理分析、生化及炎症细胞因子检测以评估胰腺损伤程度，观察PTL分解TG产生的FFAs对胰腺坏死和中性粒细胞NET形成作用。分别构建AAV-sh- CEL/PLRP1/PLRP2/PTL病毒载体，验证干扰效应后转染GPI-/-小鼠并以雨蛙肽诱导HTGP以明确关键的脂肪酶，以棕榈油酸（POA）联合酒精（EtOH）腹腔注射GPI-/-小鼠和奥利司他的干预确证PTL在HTGP重症化的作用。体外观察PTL和乳糜微粒孵育的FFAs对胰腺腺泡细胞的损伤，双重免疫荧光观察PTL的脂毒性诱导中性粒细胞死亡和释放DNA的作用。.研究结果：雨蛙肽诱导GPI-/-小鼠的胰腺组织坏死评分及总病理评分显著增高于WT小鼠、P<0.001)、胰腺组织MPO染色、TUNEL染色阳性细胞显著多于WT小鼠（P<0.001)；免疫荧光显示胰腺坏死区有大量NETs形成；雨蛙肽诱导GPI-/-小鼠胰腺组织IL-1β、IL-6和MCP-1的mRNA表达高于WT小鼠(均P<0.01）；GPI-/-小鼠胰腺组织TG和FFAs水平、腹水淀粉酶和FFAs均显著高于WT小鼠（均P<0.01）。雨蛙肽诱导GPI-/-小鼠胰腺损伤较POA联合酒精诱导GPI-/-小鼠更重，血PTL水平更高；干扰PTL的表达后能减轻了雨蛙肽诱导的胰腺损伤和坏死程度；使用PTL的抑制剂奥利司他也能减轻雨蛙肽诱导GPI-/-小鼠的胰腺组织损伤和肺损伤。体外实验发现PTL的活性与FFAs产生水平呈剂量相关性，PTL分解乳糜微粒产生的FFAs导致LDH升高和腺泡细胞坏死，并可诱导中性粒细胞死亡释放DNA。.结论：PTL是HTGP的重症化的关键胰脂肪酶，它分解血TG产生大量FFAs引起腺泡细胞损伤，诱导中性粒细胞死亡、NETs形成和DNA释放。