LncRNA MEG3/miR-21通过LAMP-2介导自噬流受阻引起程序性坏死加重急性胰腺炎分子机制研究

Severe acute pancreatitis（SAP）is a intractable acute abdominal disease with a high mortality in clinical treatment. Recent studies showed that autophagy played a significant role in AP, but the related mechanism is still unclear. In our previous studies, we found that the down-regulation of LAMP-2 in pancreatic acinar cells (PAC) can induce impaired autophagic flux when AP occured. Further cell experiments showed that the accumulation of autophagic vacuoles after impaired autophagic flux might promote the formation of necrosome to induce necroptosis. In order to investigate the mechanism of LAMP-2 down-regulation, we find significant down-regulation of MEG3 via high throughput sequencing. Bioinformatics analysis and luciferase reporter gene show that both of MEG3 and LAMP-2 can potentially combine miR-21 and downregulation of MEG3 by siRNA can upregulate miR-21 but negetively regulate LAMP-2. Therefore, we hypothesized that MEG3/miR-21 can impaire autophagy flux to induce necroptosis and exacerbate AP by down-regulating LAMP-2. To verify the hypothesis, the LAMP-2-/-, RIP3-/-and AP mice are used to check whether impaired autophagy flux damaged pancreas by inducing necroptosis, and the primary pancreatic acinar cells are used to explore the molecular mechanism of impaired autophagy flux and its induced necroptosis by co-immuneprecipitation，immunefluorescence and RIP assay to provide a new target for the clinical treatment of AP.

重症AP是临床常见急腹症，治疗困难且死亡率高。近年研究发现自噬在AP中发挥着重要作用，但具体机制不详。我们前期发现AP发生时胰腺腺泡细胞（PAC）内LAMP-2下调致自噬流受阻，进一步细胞实验发现PAC自噬流受阻后积累的自噬泡可能为necrosome的形成提供场所而诱发程序性坏死。为探究LAMP-2下调机制，我们采用高通量测序发现AP中MEG3明显下调，生物信息学分析和双荧光素酶报告基因证实MEG3、LAMP-2均可与miR-21结合，且PAC中敲低MEG3可上调miR-21而下调LAMP-2。因此，我们提出假说：MEG3/miR-21可通过调控LAMP-2介导自噬流受阻而诱发程序性坏死，加重AP。为此，本课题拟采用LAMP-2/RIP3敲除小鼠及AP小鼠模型探究自噬流受阻与程序性坏死的关系，并使用免疫共沉淀、荧光及RIP实验等在原代PAC中探究其分子机制，为AP临床治疗提供新靶点。

重症AP是临床常见急腹症，治疗困难且死亡率高。近年研究发现自噬在AP中发挥着重要作用，但具体机制不详。我们前期发现AP发生时胰腺腺泡细胞（PAC）内LAMP-2下调致自噬流受阻，进一步细胞实验发现PAC自噬流受阻后积累的自噬泡可能为necrosome的形成提供场所而诱发程序性坏死。为探究LAMP-2下调机制，我们采用高通量测序发现AP中MEG3明显下调，生物信息学分析和双荧光素酶报告基因证实MEG3、LAMP-2均可与miR-21结合，且PAC中敲低MEG3可上调miR-21而下调LAMP-2。因此，我们提出假说：MEG3/miR-21可通过调控LAMP-2介导自噬流受阻而诱发程序性坏死，加重AP。然而，受疫情影响，我们始终未获得LAMP-2敲除小鼠，但鉴于程序性坏死在AP中具有重要作用，我们将研究方向转为了胰腺腺泡细胞程序性坏死对浸润巨噬细胞的调控机制。我们使用野生型小鼠、MLKL敲除小鼠和RIPK3敲除小鼠构建了AP模型，对胰腺组织进行了相关检测，同时我们也提取了腹腔巨噬细胞和胰腺原代腺泡细胞进行体外实验，还进行了胰腺腺泡细胞的转录组测序，最终将测序结果在小鼠体内实验进行了验证。结果显示：在AP小鼠胰腺中及雨蛙素处理的胰腺原代腺泡细胞中，MLKL及其磷酸化蛋白p-MLKL显著上调，但RIPK3并无显著改变，p-MLKL的上调独立于其经典上游RIPK3。敲除MLKL可以减轻小鼠AP，但敲除RIPK3则无保护作用。有趣的是，敲除MLKL对小鼠AP的保护作用独立于细胞死亡，而主要是通过促进浸润巨噬细胞向M2极化从而减轻AP，通过高通量测序，我们发现MLKL敲除可显著降低损伤的胰腺腺泡细胞释放CXCL10，进而减少了巨噬细胞向M1极化，最后，我们发现抗CXCL10抗体可有效减轻小鼠AP的严重程度。因此，我们的研究揭示了在AP中MLKL独立于RIPK3和细胞死亡调控腺泡细胞损伤和浸润巨噬细胞极化的机制，为后续科研和临床转化提供了依据。