B细胞分泌CCL7趋化Gr1hi单核细胞肝脏浸润促进肝纤维化发生的机制研究

Currently, almost none effective anti-fibrotics for liver fibrosis has entered clinical practice. Immune responses have been demonstrated to play an important role in the pathogenesis of liver fibrosis. Data from our own laboratory have shown that B cells were increased significantly in the liver tissues of patients with chronic liver diseases and were positively correlated with the infiltration of monocytes. Meanwhile, the expression of CCL7 increased remarkably in the liver tissues of fibrotic mice. It is reported that B cells can secrete CCL7 to induce Gr1hi monocyte recruitment into the heart and result in tissue injury. Our previous studies have confirmed that Gr1hi monocytes may infiltrate into the liver tissues upon liver injury and booster the progression of liver fibrosis by releasing the pro-inflammatory and pro-fibrogenic cytokines. However, the mechanisms of monocyte liver infiltration are not yet fully elucidated. We speculate that upon liver injury, B cells may be activated and secrete CCL7 to up-regulate the expression of MMP9 in Gr1hi monocytes. These actions may promote the infiltration of Gr1hi monocytes into the liver tissues and release large amounts of pro-inflammatory and pro-fibrotic cytokines to promote liver fibrosis. To confirm this hypothesis, a series of experiments, including using B cell-deficient mouse, flow cytometric sorting, CCL7 blocking and cell migration assay will be carried out to clarify the role and mechanism of CCL7 secreted by B cells promoting liver fibrosis via inducing liver Gr1hi monocyte infiltration. The present study will provide novel targets for liver fibrosis interventions.

肝纤维化缺乏有效的治疗手段，其免疫发病机制受到重视。我们前期实验结果显示B细胞在慢性肝病患者肝组织中明显增多且与浸润的单核细胞呈正相关，而肝纤维化时肝组织中CCL7表达明显增高。文献报道，B细胞能够通过分泌CCL7介导Gr1hi单核细胞向受损的心肌浸润。我们前期研究也证实Gr1hi单核细胞在肝损伤时会趋化进入肝组织内，通过分泌促炎因子和促纤维化因子促进肝纤维化的发生，但其肝脏浸润的机制尚不清楚。我们推测：当肝组织损伤时，肝组织中的B细胞被活化，通过分泌CCL7上调Gr1hi单核细胞MMP9的表达，从而趋化其向肝脏浸润，并释放大量的促炎因子和促纤维化因子，促进肝纤维化的发生。为验证上述假说，本研究拟采用B细胞缺陷小鼠、流式细胞分选术、CCL7蛋白阻断、细胞迁移等实验揭示B细胞分泌CCL7趋化Gr1hi单核细胞肝脏浸润促进肝纤维化的机制，为寻找抗肝纤维化新靶点提供思路。

既往研究证实Gr1hi单核细胞在肝损伤时会被趋化进入肝组织内，通过分泌促炎因子和促纤维化因子促进肝纤维化的发生，但其肝脏浸润的机制尚不清楚。我们前期实验结果显示B细胞在慢性肝病患者肝组织中明显增多且与单核细胞浸润程度呈正相关，且肝纤维化时肝组织中CC趋化因子7（CCL7）表达明显增高。而在急性心肌梗死小鼠模型中，有研究发现B细胞能够通过分泌CCL7介导Gr1hi单核细胞向受损的心肌浸润。因此，我们推测当肝组织损伤时，肝组织中的B细胞被活化，通过分泌CCL7上调Gr1hi单核细胞MMP9的表达，从而趋化其向肝脏浸润，并释放大量的促炎因子和促纤维化因子促进肝纤维化的发生。为证实该假说，我们开展了以下实验：分析不同肝纤维化分期的CLD患者肝组织中B细胞、单核细胞及CCL7的差异，探讨B细胞及CCL7与肝纤维化的关系；分析CCL7对Gr1hi单核细胞肝脏浸润促进肝纤维化的作用；进一步通过体外细胞培养及动物实验，明确巨噬细胞产生的CCL7促进Gr1hi单核细胞黏附、迁移与侵袭的机制；以CCL7为干预靶点，进行改善小鼠肝纤维化模型的肝纤维化程度的体内实验。本研究通过建立小鼠肝纤维化模型证实了小鼠肝纤维化程度与Gr1hi单核细胞浸润程度呈正相关，但B细胞在肝纤维化小鼠模型中比例无明显升高；证实小鼠肝纤维化时CCL7明显升高，与小鼠Gr1hi单核细胞浸润呈正相关；体外实验证实了CCL7对单核细胞的趋化作用，同时动物实验证实抑制CCL7能减少小鼠单核细胞肝脏浸润；动物实验证实肝纤维化期巨噬细胞是CCL7分泌细胞，体外实验证实库否细胞和单核细胞均能分泌CCL7，但B细胞不是主要CCL7的分泌细胞；证实RAW264.7细胞活化为M1型巨噬细胞时，可以分泌大量CCL7，其分子机制与ERK1/2通路相关；抑制ERK1/2通路可以减少巨噬细胞分泌CCL7及对单核细胞的趋化作用。本研究充分阐述了肝纤维化时CCL7促进单核细胞肝脏浸润参与肝纤维化过程的机制，并揭示CCL7作为肝纤维化治疗靶点的可能性，本研究为肝纤维化的治疗提供新的思路。但B细胞不能通过分泌CCL7参与肝纤维化的发生，可能还存在其他机制有待进一步研究。