C/EBPa-/MITF-E+调控巨噬细胞向破骨细胞转分化消退心包钙化的作用及机制研究

Calcification is the key event contributing to the progression of a variety of pericardial diseases and degeneration of autologous pericardial repair materials. Recent studies have suggested that osteoclastic calcium resorption is a potential pathway to induce ectopic calcification regression, but unfortunately, the lack of osteoclasts in calcified pericardia restricts the application of this procedure. Given that calcified areas are surrounded by an abundance of macrophages, and the same progenitors give rise to either osteoclasts or macrophages, we hypothesize that phenotypic transcription factor-mediated transdifferentiation of macrophages into osteoclasts in situ might be an alternative to increasing the number of osteoclasts. In our previous study, we have confirmed that inactivation of C/EBPα and sustained expression of MITF-E (CM) triggered the phenotypic switch between macrophages and osteoclasts. Based on the above, the aim of the present study is to examine the mechanism underlying initiation and maintenance of CM-mediated transdifferentiation. In addition, in vitro and in vivo models will be transduced with an adenovirus vector encoding a construct overexpressing MITF-E and C/EBPα siRNA under the control of a macrophage-specific promoter, and the function of transdifferented osteoclast will be monitored. In conclusion, we will elucidate the mechanisms underlying CM-mediated transdifferentiation and examine the effects of osteoclasts transdifferentiation on pericardial calcification regression.

钙化是多种心包疾病进展和自体心包修复材料衰败的关键事件，而最新研究表明用破骨细胞吸收组织中沉积的钙是诱导异位钙化消退的一个潜在通路，但遗憾的是钙化心包缺少破骨细胞。鉴于巨噬细胞在钙化心包中分布的普遍性以及与破骨细胞起源的相近性，我们设想采用细胞表型转录因子将心包中的巨噬细胞原位转分化为破骨细胞将是增加其细胞数量的新策略。前期，我们已证实敲除C/EBPα的同时、增加MITF-E的表达（CM）可启闭巨噬细胞和破骨细胞之间的表型开关。因而我们拟以此为契机，旨在进一步分析CM启动和维持巨噬细胞和破骨细胞表型转换的分子信号调控机制；同时，构建体外和体内心包钙化模型，采用巨噬细胞特异性启动子实现CM在巨噬细胞中的特异表达，观察转分化的破骨细胞对钙盐的吸收能力。通过此研究，力图阐明CM作用机制，明确原位转分化破骨细胞消退心包钙化的可行性，并为其它钙化性疾病的防治提供理论依据。

心包钙化的消除是当今心血管领域中亟待攻克的课题和科研热点。在本项目中，我们依次明确MITF-E、C/EBPα对单核巨噬细胞/破骨细胞表型转分化的作用及其分子机制。研究结果表明：(1)在单核巨噬细胞/破骨细胞表型转分化过程中，MITF-E、TRAP表达升高。靶向抑制MITF-E后，细胞生长缓慢、多核细胞形成减少，并且TRAP的mRNA和蛋白表达降低。生物信息学分析TRAP启动子区存在MITF-E结合位点。染色质免疫共沉淀表明MITF-E蛋白能与TRAP启动子区-268～-432区域结合。荧光素酶报告基因检测则进一步表明，MITF-E能促进TRAP的转录调控，调控位点为5’-CAACAG-3’。 (2)在单核巨噬细胞/破骨细胞表型转分化过程中，C/EBPα、cathepsin K表达升高。靶向抑制C/EBPα后，细胞生长缓慢、多核细胞形成减少，并且cathepsin K的mRNA和蛋白表达降低。生物信息学分析cathepsin K启动子区存在C/EBPα结合位点。染色质免疫共沉淀表明C/EBPα蛋白能与cathepsin K启动子区-1463～-1569区域结合。荧光素酶报告基因检测则进一步表明，C/EBPα能促进cathepsin K的转录调控，调控位点为列5’-ATTGCAC-3’。 (3)钙化心包的心包间质细胞呈衰老细胞状态，高表达IL-6、MCP-1、TNF-α mRNA，能促进单核巨噬细胞的吸引和粘附。此外，衰老的心包间质细胞能沉积大量钙盐、合成胶原纤维，高表达纤维化和钙化相关基因。(4) 体外实验表明，靶向抑制MITF-E、C/EBPα后，表型转分化的破骨细胞数量减少、钙盐吸收量降低。而体内实验表明，原位注射的细胞存活率低，未检测到钙盐沉积量减少。脱细胞处理牛心包组织，则有效降低免疫原性，并减少异物反应，抑制钙盐沉积。以上结果表明：(1) MITF-E直接结合到TRAP启动子促进TRAP的转录表达进而调控单核巨噬细胞/破骨细胞表型转分化。(2) C/EBPα直接结合到cathepsin K启动子促进cathepsin K的转录表达进而调控单核巨噬细胞/破骨细胞表型转分化。(3)心包间质细胞衰老是促进单核巨噬细胞聚集的重要分子机制。(4) MITF-E、C/EBPα调控破骨细胞的吸收功能，脱细胞是减少异种心包钙盐沉积的重要方法。

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