| 摘要: | 第一型多發性神經纖維瘤 (Neurofibromatosis 1, NF1) 為最常見之神經皮膚合併症 (neurocutaneous disorder); 此症最主要特徵為患者終生好發大小深淺位置不一之皮膚內或神經內之神經纖維瘤, 因而造成嚴重之外觀缺陷或甚而影響活動. 多發性神經纖維瘤之主要致病機轉已知為NF1 基因表現之缺陷所致; NF1 乃一腫瘤抑制基因(tumor suppressor gene), 其作用為頡抗致癌基因(oncogene) RAS 所誘發之促進細胞增生以及防止細胞凋亡之現象; 因此NF1 基因表現之缺陷易造成神經纖維瘤之形成. 神經纖維瘤腫瘤本身為多種類型不同之細胞所組成, 包括Schwann cells, 肥大細胞(mast cells), 纖維母細胞(fibroblasts), 血管內皮細胞(endothelial cells) 等. 過往研究雖已指出 Schwann cells 為誘導神經纖維瘤腫瘤發生之前驅細胞, 然而針對肥大細胞(mast cells) 為何出現於神經纖維瘤腫瘤內以及其所扮演之角色究竟為何仍所知有限. 另一方面, 近年許多研究已指出, 在許多發炎性或腫瘤性疾病之病程中, 趨化激素(chemokine) 以及其受體(chemokine receptors) 對於誘導相關之發炎細胞或腫瘤細胞移行(migration or trafficking) 進入病灶部位扮演了相當重要之角色; 我們因此假設趨化激素以及其受體對於肥大細胞(mast cells) 移行進入神經纖維瘤腫瘤病灶之過程可能也有重要影響. 再者, 近期研究亦指出趨化激素受體CCR3 在肥大細胞(mast cells) 有明顯之表現, 而且對於肥大細胞種種細胞之功能, 例如細胞移行(trafficking), 細胞表型(phenotype), 以及細胞effector 功能各方面皆扮演重要之調控角色; 我們因此進一步假設Schwann cells 可能藉由CCR3 之調控, 吸引並誘導肥大細胞移行進入神經纖維瘤腫瘤之病灶. 基於以上之假設, 我們進行了多項實驗並獲致以下之結果: (1) 我們進一步證實了趨化激素受體CCR3 在我們所使用的老鼠骨髓細胞分化而來之肥大細胞(bone marrow-derived mast cells, BMMCs) 有明顯之表現, (2) CCR3 所相對應的趨化激素 ligand 之一, CCL7, 在我們所使用的Schwann cell 細胞株SW10 有明顯之表現, (3) 肥大細胞(BMMCs) 在趨化反應之實驗中顯示出對CCL7 以及培養SW10 細胞之 conditioned medium 皆有明顯之特異性之趨化反應(specific chemotaxis response), (4) 單純外加CCL7 無法增進肥大細胞之細胞存活率(cell survival) , 但是若加入CCL7 之頡抗性抗體, 卻可以明顯降低肥大細胞之細胞存活率, (5) 與纖維母細胞相較, NF1 基因於 Schwann cell 細胞株SW-10 之表現較低. 基於上述研究結果, 我們有以下結論: (1) Schwann cells 可以藉由CCR3 與CCL7 之交互作用吸引肥大細胞, 這可能可以作為誘導肥大細胞移行進入神經纖維瘤腫瘤病灶的機轉之一, (2) 雖然外來(exogenous) 之 CCL7 (paracrine) 對於肥大細胞之趨化性以及細胞移行可能十分重要; 如何進一步維持肥大細胞於神經纖維瘤腫瘤病灶內之存活可能有賴於肥大細胞自身分泌之CCL7 (autocrine), (3) CCR3 與CCL7 所調控之肥大細胞移行現象, 與相關細胞如Schwann cells 致病基因NF1 表現之缺陷可能亦有相關性. 雖然我們已經獲致上述研究成果, 然而關於神經纖維瘤相關致病機轉以及治療發展之相關問題仍有許多問題尚待探討研究, 我們於此提出以下三個主要方向做為此研究計畫之主軸: (1) 首先我們將嘗試利用RNA interference 技術進一步干擾NF1 基因於 Schwann cell 細胞株SW10 以及肥大細胞中之表現; 我們進而將嘗試利用這些缺乏NF1 基因表現之SW10 細胞以及肥大細胞注射進入同種老鼠皮下或靜脈以期能建立模擬皮膚神經纖維瘤之動物模式; (2) 其次, 我們將進一步探討趨化激素以及其受體, 例如 CCL7/CCR3, 是否在肥大細胞由週邊血流穿過血管而進入組織這一段移行過程中, 也扮演了重要之調控角色; 我們也計畫進一步研究這段過程是否與其他發炎細胞由血流進入組織之過程類似,也歷經integrin 之活化與黏著等順序與步驟; (3) 此外, 由於目前神經纖維瘤之治療選擇仍極端缺乏; 我們也將嘗試根據動物模式以及肥大細胞移行機轉之研究結果, 進一步選擇特定分子, 例如可能在肥大細胞移行過程中扮演重要角色之CCR3 或是4 integrin, 並利用這些分子之頡抗性抗體進行動物實驗; 以其進一步開發可用於神經纖維瘤治療之新策略. 我們認為本計畫將對於神經纖維瘤之致病機轉之進一步了解, 以及治療方法之開發; 做出可觀之貢獻
Neurofibromatosis 1 (NF1) is the most common neurocutaneous disorder featured by multiple neurofibromas which often cause life-long suffering of severe disfiguring and loss of function in patients. NF1, a tumor suppressor gene able to inhibit RAS-mediated cell proliferation and apoptosis-resistance, is deficient in NF1. Characterized by cellular heterogeneity, neurofibroma is composed of many types of cells including Schwann cells (SCs), mast cells (MCs), fibroblasts, endothelial cells, and perineural cells. While SCs have been established as the major progenitor cells for neurofibroma formation, our knowledge about how MCs become one of the constituent cells of the tumor and whether they may also contribute to neurofibroma tumor formation and progression remains limited. As chemokines and chemokine receptors have been well established to be essential for the migration of inflammatory or malignant cells to the lesions of many inflammatory diseases or cancers, we hypothesize that chemokine and chemokine receptors may also play a role in the mast cell migration to the neurofibroma. Since recent reports have shown that CCR3 plays multiple essential roles in MCs, including trafficking of MC progenitors and selective modification of MC phenotype and effector function, we hence further hypothesize that SCs may attract MCs by CCR3-mediated mechanisms. We then performed several series of experiments and have found some major results as follows : (1) expression of CCR3 by mouse bone marrow-derived MCs (BMMCs) has been confirmed, (2) one of CCR3 ligands, CCL7, is expressed by mouse SC line SW10, (3) BMMCs were specifically responsive to chemokine CCL7 and conditioned medium from SW10 cells, (4) while exogenous CCL7 could not increase BMMCs’ survival, it was significantly decreased by CCL7-neuralizing mAb, and (5) the expression of pathogenic gene NF1 in SW-10 is lower in comparison to mouse fibroblast. Based on the above results, we have concluded that: (1) SCs may induce MC migration by interaction between CCR3 and CCL7, which may in part explain the presence of many MCs in neurofibroma, (2) while exogenous (paracrine) CCL7 may be important for MC chemotaxis and migration, MC survival may depend on autocrine CCL7 secretion, and (3) possible correlation may exist between NF1 gene deficiency (pathogenic for neurofibroma) and CCR3-mediated MC migration. Although we have obtained these substantial results, there are still many questions left to be addressed in the pathogenesis and treatment of neurofibroma and we propose to continue the investigation toward three major directions as follows. First of all, we will seek to knock down the NF1 gene in SW10 cells and also MCs by RNA interference, and then we will try to establish a novel syngenic transplant animal model for cutaneous neurofibroma by using the gene-modified NF1-deficient SW10 and MCs. Secondly, we will seek to address how MC progenitors in the peripheral blood may migrate through endothelial cells and home to neurofibroma, and whether chemokine system (like CCR3 and CCL7) may play important role in the trans-endothelilal migration process through current paradigm for leukocyte adhesion cascades composing of tethering, rolling, integrin activation, and firm adhesion to endothelial cells. Serial chemokine-related experiments including adhesion activation assay, integrin adhesion assay, and trans-endothelial migration assay will be performed. Thirdly, it will be important to see whether many promising therapeutic agents, especially those targeting mast cells migration pathway (e.g. anti-CCR3 or anti-4 integrin), will be useful in vivo in the context of neurofibroma. We believe this project may contribute greatly to our further understanding of pathogenesis and treatment of neurofibroma, and may possibly develop some novel therapeutic strategies for neurofibroma. |
