巨噬细胞极化通过调控PSC活化对慢性胰腺炎胰腺纤维化的影响及大柴胡汤对其干预机制研究

Activation of pancreatic stellate cells (PSC) is the central event of the pancreatic fibrosis in chronic pancreatitis (CP). In recent years, the studies found PSC activation accompanied large numbers of macrophages (Mφ) infiltration in pancreatic tissue during the development of CP. What is the role of these macrophages in PSC activation and pancreatic fibrosis?The latest research shows that macrophage polarization can be seen in the pancreatic fibrosis. The numbers of M2 macrophages is proportional to pancreatic fibrosis, but the influence and mechanism of macrophages polarization on pancreatic fibrosis is not known. We intend to transfuse the different polarization of macrophage(M1 macrophage and M2 macrophage) labeled with DIL to SCID mice from tail vein directly and use the co-culture system of macrophages and PSC, as well as the techniques of flow cytometry and Luminex to explore the role of macrophage polarization in the activation of PSC and pancreatic fibrosis, to search the key targets induced PSC activation. We hope to verify the theory that the regulation of macrophage polarization is a measure to improve pancreatic fibrosis and provide a new strategy for the prevention and treatment of CP. .Furthermore, Dachaihu Decoction is a classic prescription for restraining inflammatory response. The effect of Dachaihu Decoction on pancreatic fibrosis is confirmed, but its mechanism is unclear. By vivo and vitro experiments, we intend to research whether Dachaihu Decoction regulate the polarization of macrophages or promote polarized macrophages transformed well, thereby reducing PSC activation and inhibiting pancreatic fibrosis. We hope to elucidate the molecular mechanism of Dachaihu Decoction treating pancreatic fibrosis, and provide the support for clinical application of Dachaihu Decoction in CP.

胰腺星状细胞（PSC）活化是慢性胰腺炎（CP）胰腺纤维化的核心事件，近来的研究发现，PSC活化的同时伴随胰腺大量巨噬细胞（Mφ）浸润。最新研究表明：Mφ在CP进展中发生了极化偏移，且与胰腺纤维化相关，但是极化的Mφ对胰腺纤维化的确切影响和调控机制尚不清楚。拟采用Dil标记的不同极化状态Mφ尾静脉注射回输至SCID小鼠体内，及Mφ-PSC非接触性共培养体系，结合流式细胞、Luminex等技术，研究不同极化状态Mφ对PSC活化的调控作用，及对胰腺纤维化的影响，明确致PSC活化的“Mφ主凶”，阐明“通过调控Mφ极化稳态改善胰腺纤维化”的理论，为CP防治提供全新策略。大柴胡汤治疗CP疗效确切，但抗胰腺纤维化的机制不清。拟通过在体和离体实验，研究大柴胡汤是否通过调控Mφ的极化稳态，进而减轻PSC活化、抑制胰腺纤维化，阐明大柴胡汤防治胰腺纤维化的分子机制，为其在CP的临床应用提供实验依据。

胰腺星状细胞（PSC）活化是慢性胰腺炎（CP）胰腺纤维化的核心事件，近年研究发现PSC活化的同时胰腺可见大量巨噬细胞（Mφ）浸润，且会发生极化偏移，不同极化状态的Mφ对PSC活化、胰腺纤维化的影响和具体机制尚不清楚。本课题分离小鼠的骨髓前体细胞、成功诱导为M1、M2型巨噬细胞，并采用PEI-SPIO/Cy3-miRNA复合物标记Mφ，将标记的不同极化状态Mφ经尾静脉注射回输至SCID慢性胰腺炎小鼠体内，研究不同极化状态Mφ对慢性胰腺炎胰腺纤维化的影响。进一步建立了Mφ-PSC共培养体系，结合流式细胞、Luminex等技术，研究不同极化状态Mφ对PSC活化的调控作用。另外，还观察了大柴胡汤对慢性胰腺炎胰腺纤维化的治疗作用，及其含药血清对PSC活化和巨噬细胞极化状态的影响，探索大柴胡汤防治CP的作用及机制。该课题还进一步探究了活化的PSC对巨噬细胞的活化和极化状态的影响，研究了大柴胡汤的主要成分黄芩苷是否通过抑制小鼠胰腺星状细胞和巨噬细胞的活化、改善胰腺纤维化。.项目的研究结果显示：不同极化状态Mφ回输后对胰腺炎症和纤维化程度有不同影响，M2型Mφ回输可以促进胰腺的炎症和纤维化，FN的表达明显升高，而M1型Mφ回输组胰腺的炎症和纤维化程度明显减轻。M2型Mφ条件培养基可以促进PSC增殖和迁移；而M1型Mφ条件培养基则可以抑制PSC增殖和迁移，这一结果表明“通过调控Mφ极化稳态可以改善胰腺纤维化”，可以作为CP防治的策略之一。通过在体和离体实验显示，大柴胡汤不仅可以减少Mφ浸润、抑制PSC活化，还可以遏制Mφ的不良极化，进而减轻PSC活化、抑制胰腺纤维化。大柴胡汤的主要成分黄芩苷既可抑制PSC分泌MCP-1来遏制巨噬细胞的迁移还可通过调控PSC中的TGF-β1/TAK-NF-κB通路发挥防治胰腺纤维化的作用。.本课题从整体-细胞-分子层面探究了不同极化状态巨噬细胞对PSC活化、胰腺纤维化的影响，及大柴胡汤遏制胰腺纤维化的分子机制，为胰腺纤维化防治提供新的思路，为大柴胡汤用于临床防治慢性胰腺炎提供科学依据。