高甘油三酯在高甘油三酯性胰腺炎胰管黏膜屏障损伤中对MLCK调控作用及其机制研究

The incidence of hypertriglyceridemia-associated pancreatitis(HTGP) has been increased in recent years. Myosin light chain kinase is a key protien which regulate the tight junction between endothelial cells. Our previous study found that MLCK was activated in long term hypertriglyceridemia, the tight junction was destroyed, the pancreatic ductal macosal barrier was damaged finally, and then sever acute pancreatitis was induced by a light triggering. Thus, we hypothesized that MLCK plays a important role through regulated by HTG in PDMB of HTGP. The primary aim of this study was: ①Establish HTG rat in high fat feed, and inducing the HTGP rat by caerulein, inhibit the activasion of MLCK with the specific inhibitor (ML-7, ML-9), then PDMB damage, the expression of MLCK, calcium channels, and the TJ associated proteins were measured in order to observe the relationship of TG and PDMB disfunction, MLCK, Ca2+, and TJ associated proteins in HTGP. ②Primary culture rat pancreatic ductal epithelial cells, regulated MLCK by genetic engineering technology. Observe the TJ associated proteins and calcium channels in the different MLCK expression cells in different triglyceridemic medium. This project may reveal the mechanism of MLCK regulated by HTG in the disfunction of pancreatic ductal macosal barrier in HTGP.

近年来高甘油三酯性胰腺炎（HTGP）发病率增高，但其发病机制未明。肌球蛋白轻链激酶（MLCK）是调节细胞紧密连接（TJ）的关键蛋白。我们研究发现，长期高甘油三酯血症（HTG）使MLCK活化，胰管上皮细胞TJ减弱，胰管黏膜屏障（PDMB）受损，导致轻微诱因下即可引起SAP，因此推测HTG可能通过调控MLCK在HTGP的PDMB损伤中发挥作用。本项目拟采用：①高脂喂养法建立HTG大鼠后以雨蛙肽诱导HTGP，应用MLCK抑制剂干预，检测大鼠PDMB、MLCK、钙通道及TJ相关蛋白表达，了解HTGP大鼠TG水平与PDMB损伤、MLCK、Ca2+以及TJ相关蛋白的关系；②原代分离培养大鼠胰腺导管上皮细胞作为模型，并采用基因工程技术改变MLCK的表达，检测不同TG水平在不同MLCK表达的细胞间TJ及其相关蛋白、钙通道蛋白的变化。以期深入探讨HTG在HTGP的PDMB损伤中对MLCK的调控机制。

目前认为，肌球蛋白轻链激酶（MLCK）是调节细胞紧密连接（TJ）的关键蛋白，推测高甘油三酯（HTG）可能通过调控MLCK在高甘油三酯性胰腺炎（HTGP）的胰管黏膜屏障（PDMB）损伤中发挥作用。本研究发现：（1）建立HTGP大鼠模型并用ML-7干预后，①HTGP组血清AMY含量、胰腺病理评分、MLCK、Cav1.2、E-cadherin、β-catenin的表达均较AP组显著升高；ML-7干预后上述指标有所改善。②透射电镜下可见HTGP组胰腺细胞超微结构损伤及TJ增宽明显；ML-7干预后TJ增宽减小。（2）体外实验：①用油酸（OA）和软脂酸（PA）与人胰腺导管上皮细胞（HPDE6C7）共培养，发现OA和PA均可抑制细胞增殖，上调MLCK及钙通道相关蛋白mRNA表达，减少TJ相关蛋白mRNA表达。②不同浓度甘油三酯（TG）与HPDE6C7细胞共培养，发现HTG可抑制细胞增殖及促进细胞凋亡，上调细胞MLCK、ICAM-1、β-Catenin、CDH-1基因表达，下调DSC-3基因表达。③雨蛙肽（CAE）与PA共培养，细胞内Ca2+浓度升高，MLCK表达增多，微丝结构破坏，TJ间隙增宽，TJ相关蛋白表达减少，细胞通透性增高，ML-7可使上述变化改善；沉默MLCK基因表达后，细胞通透性下降，TJ相关蛋白表达上调。因此，本研究认为：（1）MLCK可能通过改变细胞间TJ、粘附连接及调控Cav1.2的表达参与HTGP的发病机制。（2）①HTG具有细胞毒性作用，可改变MLCK及TJ相关蛋白的基因表达，相互协同作用引起PDMB损伤。②HTGP细胞模型中，钙超载可能使MLCK活化，进而影响细胞骨架及细胞间TJ，参与HTGP的发生发展。