miRNA551b-5p在重症急性胰腺炎毛细血管渗漏综合征中的机制研究

Capillary leakage syndrome is the intermediate stage from systemic inflammatory response syndrome to multiple organ failure in the patients of severe acute pancreatitis . The treatment of capillary leakage syndrome has very important to the patients of severe acute pancreatitis. Our previous studies have found that the lever of miRNA551b-5p in the patients of severe acute pancreatitis is higher than the patients of mild acute pancreatitis.The transcellular pathway and the paracellular pathway are the main mechanisms of capillary leakage syndrome. The transcellular pathway is mainly related to aquaporin protins.Paracellular pathway is mainly mediated by cellular junctions. We had found that miRNA associated with acute pancreatitis was related with tight and adhesive junctions and aquaporin protins by the genes of EGFR yet.Therefore, we intend to expand the number of samples to further confirm the relationship between miRNA551b-5p and capillary leakage syndrome. Combineing with sequencing we research the molecular mechanism of capillary leakage syndrome at the cellular and biological individual levels by up-regulating and down-regulating miRNA551b-5p. The function and mechanism of EGFR gene were screened and verified by two color fluorescence complementary verification techniques, especially the role of EGFR gene in capillary leakage syndrome. The study will help us to understanding the function of these genes related miRNA551b-5p in the mechanism of capillary leakage syndrome. The results of the study will provide a new way for people to further understand the pathogenesis and treatment of capillary leakage syndrome in the patients of severe acute pancreatitis .

毛细血管渗漏综合征(CLS)是重症急性胰腺(SAP)全身炎症反应综合症向多器官功能衰竭的中间阶段，治疗CLS对改善SAP的预后有重要的临床意义。我们前期发现了SAP相关性miRNA（miRNA551b-5p），通过预实验，观察到它会引起内皮细胞的通透性变化。其中跨细胞途径及细胞旁途径是CLS的主要机制，跨细胞途径与水通道蛋白有关，细胞旁途径与细胞连接有关，我们还发现急性胰腺炎相关性miRNA通过EGFR与内皮细胞通透性相关。因此，我们拟扩大样本量，进一步证实miRNA551b-5p与CLS的关系；通过上调及下调miRNA551b-5p，研究其在细胞水平和生物个体水平中对CLS影响的分子机制，并结合测序、荧光素酶等技术，对相应基因功能及作用机理筛查和验证，特别是EGFR基因在CLS的的作用，以了解这些基因的功能在CLS中的机制。为人们认识在SAP中发生CLS的机制及治疗提供新思路。

重症急性胰腺炎（SAP）病死率高，其急性反应期常伴有毛细血管渗漏综合征（CLS）。CLS是SAP全身炎症反应综合症向多器官功能衰竭的中间阶段，治疗CLS对改善SAP的预后有重要的临床意义。SAP并CLS与毛细血管内皮损伤有关，毛细血管内皮细胞的损伤影响毛细血管内物质转运(跨细胞途径及细胞旁途径的异常)。近年研究发现miRNA与胰腺疾病的发生、发展及预后密切相关，我们前期发现与SAP并发症发生相关的miRNA551b-5p。因此，本研究通过研究mircoRNA551b-5p在细胞和生物个体水平中对SAP并CLS影响的分子机制，并结合测序、荧光素酶等技术，探究miRNA551b-5p在SAP并CLS中的作用机制。结果显示：1.通过雨蛙素(Cre)联合脂多糖(LPS)构建SAP小鼠模型，发现相比对照组小鼠，SAP组小鼠胰腺组织的miR551b-5p显著升高（p<0.05），SAP组的胰腺及肺组织毛细血管渗漏显著增加（p<0.05）。2.使用瞬时转染，过表达及下调HUVEC的miR551b-5p，再运用免疫荧光、荧光黄Transwell、免疫荧光、WB以及Qpcr实验，结果发视miR551b-5p inhibitior组miR551b-5p下调不明显；而相比对照组，miR551b-5p mimics组的通透性显著增加，细胞骨架形成空洞，AQP5、occludin、JAM3和Claudin1的mRNA和蛋白表达显著下调；3. 构建AAV8-miR551b-5p及AAV8-miR551b-5p spone，腹腔注射至小鼠3周后再构建SAP小鼠模型，发现同细胞实验一样， miR551b-5p无明显下调；而miR551b-5p可以加重SAP组小鼠病情，降低AQP5、occludin、JAM3和Claudin4的mRNA和蛋白表达。4.经过转录组测序和生物信息学分析，发现miRNA551b-5p在SAP并CLS中可能是通过靶向ERBB3，影响PI3K/AKT信号通路，并通过QPCR和荧光素酶验证。基于以上结果，我们认为miRNA551b-5p在SAP并CLS的作用机制是：miRNA551b-5p可以通过靶向ERBB3，影响PI3K/AKT信号通路，引起AQP5、Occludin、Claudin1和JAM3表达下降，从而改变毛细血管内皮通透性，加重或引起SAP的毛细血管渗漏。