大黄附子汤对实验性重症急性胰腺炎肠黏膜上皮细胞线粒体损伤调控作用的研究

Severe acute pancreatitis (SAP) is a common acute and serious disease. In the course of SAP,enterogenic infection caused by intestinal epithelium cells(IEC) damage and intestinal barrier functional dysfunction is the significant cause giving rise to SIRS,MODS or MOF. Mitochondria (Mt) is the "productivity center","metabolic center" and "apoptosis center" of cell life. It decides the IEC's "life or death". The experimental and clinical studys have showed that Dahuangfuzi decoction can improve intestinal function of SAP,while reducing the damage of IEC-Mt. It also can keep SAP off SIRS-MODS. But the specific mechanism is not clear. This subject will reveal IEC-Mt damage change rule and the key targets and mechanism of the regulation function of Dahuangfuzi decoction in vivo and vitro aspects, learning the structure and function of equal emphasis, taking the entry point of Dahuangfuzi decoction's therapeutical effect, with IEC-Mt change as the main line,in vitro studies，detecting expression of DNA, MPTP, reactive oxygen species and apoptosis inducing factor in the different periods of the IEC-Mt in rats through fluorescent quantitative PCR and laser scanning confocal microscope et al,and using living cells workstation and flow cytometer to measure change of morphological parameters, respiratory function and ATP in vitro studies,the aim of which is to provides a new approach for the prevention and control of SAP.

重症急性胰腺炎（SAP）是常见的急危重病，SAP时肠黏膜上皮细胞（IEC）损伤及肠功能障碍引起的肠源性感染，是引发SIRS、MODS或衰竭的重要原因。线粒体(Mt)是维持细胞生命的"产能中心"、"代谢中心"和"凋亡中心"，决定着IEC的"生死"。前期实验和临床研究表明,大黄附子汤改善SAP肠道功能,减轻IEC-Mt的损伤,阻止SAP向SIRS-MODS进展,但具体机理不清。本课题以大黄附子汤治疗SAP为切入点,SAP时IEC-Mt改变为主线，体内研究应用荧光定量PCR、激光共聚焦显微镜等检测大鼠不同时段IEC-Mt的DNA、MPTP、活性氧基、凋亡诱导因子等的表达；体外研究应用活细胞工作站、流式细胞仪等动态检测IEC-Mt的呼吸功能、形态学参数、ATP浓度等变化,结构与功能学研究并重，共同揭示SAP时IEC-Mt损伤变化规律及大黄附子汤调控作用的关键靶点和机制，为SAP的防治提供新思路。

重症急性胰腺炎（SAP）是临床常见急危重症，常继发的肠黏膜上皮细胞（IEC）损伤及肠功能障碍并引起肠源性感染，后者是其导致全身炎症反应综合症（SIRS）、多器官功能不全综合症以及死亡的重要原因。肠黏膜上皮细胞线粒体在SAP后肠黏膜上皮细胞损伤中起重要作用。本研究通过大黄附子汤(DHFZT)治疗SAP后肠黏膜上皮损伤为主线，应用多种分子生物学技术，从线粒体通透性转换孔（MPTP）、ATP、细胞色素C氧化酶活性、线粒体超微结构和离子泵等方面进行了肠黏膜上皮细胞线粒体损伤的研究工作。主要研究总结如下：（1）经胰胆管注入5％牛磺胆酸钠成功建立SAP大鼠模型，SAP后引发IEC线粒体离子泵Na+-K+-ATP 酶、Ca2+-ATP酶活性显著下降，IEC及其线粒体超微结构损伤，使血清肠脂肪酸结合蛋白（iFABP）、二胺氧化酶（DAO）水平升高，DHFZT可通过升高IEC线粒体离子泵Na+-K+-ATP 酶、Ca2+-ATP酶活性，维护IEC及其线粒体超微结构，保护肠黏膜屏障，降低了肠道损伤特异性指标血清iFABP、DAO的水平。（2）SAP后大鼠肠黏膜上皮细胞线粒体ATP合成酶和细胞色素C氧化酶活性均显著下降，细胞能量代谢和呼吸功能出现障碍，大黄附子汤能通过增强大鼠肠黏膜上皮细胞线粒体ATP合成酶和细胞色素C氧化酶活性，保护SAP后肠黏膜上皮细胞的损伤，降低了血清内毒素含量，减轻SAP病情。（3）大黄附子汤能通过降低SAP大鼠肠黏膜上皮细胞线粒体通透性转换孔（MPTP）的开放，增加线粒体ATP的生成量，增强细胞色素C氧化酶活性，减轻了肠黏膜上皮细胞损伤，从而显著降低了大鼠血清中淀粉酶、内毒素、DAO、D-乳酸的含量。这些研究共同揭示了大黄附子汤对SAP时肠粘膜上皮细胞线粒体损伤保护作用的关键靶点和机制，为大黄附子汤临床应用以及后期开发、推广奠定理论基础。已发表学术论文3篇，被SCI收录1篇（另1篇SCI论文外投中）。培养硕士1名，已毕业。