Reg4在急性胰腺炎后胰腺再生中的作用及分子机制研究

The mortality of severe acute pancreatitis (SAP) remains high, as lack of effective drugs to cure SAP. Pancreatic inherent ability to resist damage and self-repair is one of most important determinants influencing the prognosis of acute pancreatitis (AP). Our preliminary study showed that Reg4 expression was upregulated and then declined gradually down to normal during pancreatic injury and regeneration phase after acute pancreatitis. Expression of Reg4 was mainly localized in islet, residual acinar cells nearby islet and regenerating metaplastic epithelium. Recombinant human Reg4 (rReg4) protein ameliorated the mortality rate and the severity of experimental AP induced by both arginine and caerulein in vivo. In addition, rReg4 directly protected pancreatic acinar cells from necrosis induced by arginine in vitro. Furthermore, conditional knock out of Reg4 gene exacerbated pancreatic damage induced by caerulein and suppressed regeneration of exocrine pancreas after induction of AP. These results indicated that Reg4 probably participate in the regeneration of pancreas after acute pancreatic damage. In this study, the exact role of Reg4 in acinar cell proliferation, dedifferentiation and regeneration during experimental AP will be explored using gene-knock out technique and small interfering RNAs. Mechanism of Reg4 in regulating EGFR and Notch signaling will also be elucidated through overexpression of Reg4 gene and applying rReg4 protein. These results will probably help unravel the molecular pathogenesis of pancreatic regeneration and provide insight into the development of novel therapeutic agent for pancreatic diseases.

重型急性胰腺炎（SAP）尚乏理想的药物治疗，其死亡率较高。胰腺内在的抗损伤和再生能力是影响急性胰腺炎（AP）预后的重要因素之一，但胰腺再生的分子机制远未阐明。本课题组前期发现在小鼠AP后损伤和修复模型中Reg4基因和蛋白表达呈先上升后下降的趋势，免疫荧光发现Reg4的表达主要分布在胰岛和胰岛周围残存的腺泡细胞及新生导管样上皮细胞中；使用重组Reg4蛋白减轻了AP的严重程度，而Reg4基因条件敲除加重胰腺损伤并明显抑制了胰腺再生，提示Reg4可能参与AP后胰腺抗损伤和再生过程。本研究拟利用基因敲除、RNAi技术研究Reg4对AP后腺泡细胞增殖、去分化及再分化的影响，以明确Reg4在胰腺再生过程中的作用，并通过高表达Reg4和应用外源性重组Reg4蛋白观察其对胰腺腺泡细胞EGFR和Notch信号通路的调节机制。本研究的完成将进一步阐明胰腺再生的分子机制并为胰腺功能障碍相关疾病治疗提供新的思路。

重症急性胰腺炎目前尚无有效的药物治疗，有很高的死亡率。同时，重症胰腺炎患者在急性炎症期后极有可能存在因胰腺再生不全所引起的胰腺外分泌功能不全，从而引起骨质疏松等更加严重的并发症并极大的影响患者的生存质量，但目前对胰腺炎后胰腺再生的机制尚不十分明确。我们发现，Reg4蛋白在胰腺炎期间的表达存在上调，且将其敲除之后严重影响了胰腺组织的再生修复。我们进一步在Reg4敲降及再生期特异性Reg4敲降小鼠模型上验证了这一现象，说明Reg4在胰腺再生过程中发挥作用。我们进一步对其作用机制进行探究发现，Reg4敲除及敲降均减少了胰腺再生期间正常腺泡细胞的区域，但免疫组化/免疫荧光染细胞增殖标记物Ki-67的结果显示Reg4敲降后反而增加了处于增殖状态的腺泡细胞数量。同时我们通过免疫荧光共染淀粉酶及CK19/SOX9发现，Reg4敲降的胰腺组织中存在着腺泡细胞导管化生结构的增多及持续存在，体外腺泡细胞的3D培养也发现Reg4敲降的腺泡细胞更快的形成了更多的导管化结构。以上结果说明Reg4通过影响腺泡细胞的去分化及再分化延缓了胰腺炎后胰腺再生。此外，我们对腺泡细胞分化过程中的关键干性信号通路进行了分析，发现Reg4敲降小鼠的胰腺组织中存在NOTCH信号通路的过度激活，说明Reg4可能通过调节NOTCH信号通路影响了腺泡细胞去分化及胰腺再生。我们的研究揭示了Reg4在胰腺炎后胰腺再生中的作用及其相关分子机制，为急性胰腺炎患者再生期的的治疗提供了理论依据，同时为后续急性胰腺炎后胰腺再生的研究开辟了新的领域并提供了新的手段。