内质网应激诱导自噬对重症急性胰腺炎淋巴细胞凋亡的作用及分子机制

Severe Acute Pancreatitis (SAP) is a common severe disease and its underlying mechanisms are not fully understood. Aggressive inflammatory immune response has been reported at the early stage of SAP and immunosuppression at later stage of SAP. The results of our previous study showed that the lymphocyte apoptosis is one of the important reasons of immunesuppression in SAP, but its mechanism is unclear. It has been reported that the autophagy induced by endoplasmic reticulum stress (ER stress) was involved in the damage of pancreatic acinar cells, but there is no research based on relationship between autophagy induced by ER stress and lymphocyte apoptosis of SAP. In order to figure out their relationship and the underlying mechanisms, the experiments are performed. Autophagy, apoptosis and endoplasmic reticulum of lymphocyte are detected by using transmission electron microscopy and flow cytometry. Expression of related genes of autophay and apoptosis in IRE-1 and PERK signaling pathways of ER stress are detected by using the methods of fluorescence quantitative PCR and western blot. At the same time, the lymphocyte in peripheral blood of SAP rats is extracted and culture. Upstream promoter genes, IRE-1 and PERK, in the signal pathways of ER stress are silented by using RNA interference technique. Autophagy, apoptosis , endoplasmic reticulum of lymphocyte and expression of related genes in IRE-1 and PERK signaling pathways of ER stress are detected again. This study was designed to investigate the role of autophagy induced by ER stress in lymphocyte apoptosis of SAP and the underlying mechanisms. So as to lay the theoretical basis for immune intervention treatment in clinic.

重症急性胰腺炎（SAP）是临床常见的危急重症，然其发病机制未明。研究表明SAP早期炎症免疫反应过激，后期则出现免疫抑制。在前期研究中，我们发现淋巴细胞大量凋亡是引起SAP免疫抑制的重要原因，但其发生机制尚不清楚。已有研究表明，内质网应激诱导自噬介导了胰腺腺泡细胞损伤，但未见有在SAP时是否参与淋巴细胞凋亡的研究报道。本课题在前期研究基础上，采用透射电镜观察SAP大鼠淋巴细胞自噬、凋亡及内质网超微结构，流式细胞术检测自噬率、凋亡率，RT-PCR、Western blot等技术研究内质网应激IRE-1、PERK信号通路中自噬、凋亡相关基因mRNA及蛋白表达；并通过体外培养大鼠外周血淋巴细胞，采用RNA干扰技术靶向沉默信号通路中的上游启动基因IRE-1及PERK，观察上述指标的变化，试图明确内质网应激诱导自噬对SAP淋巴细胞凋亡的作用及可能的分子机制，为临床进行免疫干预治疗提供理论基础。

淋巴细胞大量凋亡是引起重症急性胰腺炎（SAP）免疫抑制的重要原因，但其发生机制尚不清楚。本项目采用透射电镜观察 假手术组、SAP 组、3-MA干预组大鼠淋巴细胞自噬、凋亡、内质网超微结构，流式细胞术检测自噬率、凋亡率，RT-PCR、Western blot 等技术研究内质网应激 IRE-1、PERK 信号通路中自噬、凋亡相关基因 mRNA 及蛋白表达。结果发现：SAP 时 ER stress（内质网应激）信号途径激活促进淋巴细胞自噬、凋亡，当自噬抑制时淋巴细胞凋亡增强，ER stress 诱导自噬参与SAP 淋巴细胞凋亡的调控，IRE-1、PERK、Beclin1、Atg8/LC3、Bax、Caspase-3是参与调控的关键因子。我们还通过体外培养大鼠脾淋巴细胞，采用 RNA 干扰技术靶向沉默 ER stress 信号通路中的上游启动基因 IRE-1 及 PERK，观察淋巴细胞自噬、凋亡及信号通路中上述因子的变化，结果IRE-1 及 PERK 沉默后淋巴细胞自噬、凋亡减少，Beclin1、Atg8/LC3、Bax、Caspase-3 表达下降，进一步确认了 ER stress 信号途径对SAP淋巴细胞自噬、凋亡的影响及参与调控的分子机制，PERK 及 IRE-1可能作为调控靶点。本项目已发表论文5篇，其中中文核心期刊论文2篇，待发表论文3篇。另外，本项目基金资助培养硕士研究生5人，其中已毕业2人；项目负责人入选第二批广西医学高层次骨干人才培养“139”计划中青年学科骨干人才培养人选，并成为校级学术带头人。

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