巨噬细胞活化对先天性巨结肠Cajal间质细胞表型改变的影响

The phenotype change of the intersitial cells of Cajal (ICCs) takes important role in the pathogenesis of Hirchsprung's disease (HD), but the underlying mechanisms are still unclear. This project is based on our previous study and to investigate the differential macrophages activation and c-kit protein expression in HD animal model with and without entestitis, and to find out the association among macrophage activation, TNF-α secretion, and the phenotype change of ICCs. In addition, it is to find out the effect of TNF-α on the expression of c-kit and CD34 in ICCs in vitro, and to directly observe the phenotype change of ICCs on TNF-α exposure. Furthermore, it is to investigate the effect of TNF-α on c-kit and CD34 of ICCs in miR221 and miR222 suppressed zerbrafish intestine, of which exhibits higher miRNA inhibition efficiency and has the advantage in directly observing intestinal transit time than mice model, to understand the causal relationship within HD intestinal macrophage activation → TNF-α secretion → miRNA increase → inhibition of c-kit expression → phenotype change of ICCs → impairment in intestinal movement. Our project is to lay the theoretical foundation to reverse the phenotype change of ICCs and further improve the HD treatment effect.

Cajal间质细胞表型改变在先天性巨结肠的发病中起重要作用，但机理不明。本项目在前期研究基础上，探讨HD动物模型肠炎与非肠炎组及对照组中巨噬细胞活化和c-kit表达的改变，然后抑制巨噬细胞活化，观察巨噬细胞活化、分泌TNF-α与ICCs表型改变间的关系。同时研究TNF-α对体外培养ICCs之c-kit和CD34表达的影响，以直接观察TNF-α引起ICCs表型改变。继而采用新型模式生物斑马鱼作为研究对象以弥补小鼠miRNA抑制效率低和不能直接观察肠道传输时间的不足，抑制斑马鱼miR221、222后再观察TNF-α对斑马鱼肠道c-kit和CD34表达及肠道传输的影响，从而弄清HD肠壁巨噬细胞活化→分泌TNF-α→增加miRNA→抑制c-kit表达→ICCs表型改变→肠道运动受损间的因果关系，为逆转ICCs表型改变，进一步提高HD的治疗效果奠定理论基础。

Cajal间质细胞发育异常在先天性巨结肠的发病中起重要作用，前期有研究表明，在人类先天性巨结肠中，结肠远段无神经节段Cajal间质细胞减少，并且在有神经节段Cajal间质细胞分布亦有异常，但具体机理不清。本研究中，我们采用EdnrB基因敲除小鼠作为研究先天性巨结肠研究的模型动物，收集1w，2w，3w的巨结肠小鼠及野生型小鼠结肠，并依据部位分为远端、中段、近段结肠。PGP9.5及NeuN免疫荧光双染证实了EdnrB基因敲除小鼠远端结肠神经节细胞缺失；HE染色表明，3w巨结肠小鼠结肠中段及近段扩张段炎症浸润；同时采用CD68及iNOS免疫荧光双染证实了巨结肠小鼠扩张段主要以经典活化的巨噬细胞细胞浸润为主，免疫组化染色表明Cajal间质细胞在扩张段明显减少，并且WB及RT-PCR证实TNF-α与c-kit表达呈现负相关关系；电生理记录仪检测到3w巨结肠小鼠肠道慢波破坏。随后我们采用氯膦酸二钠脂质体清除巨噬细胞后，观察到3w巨结肠小鼠结肠中段及近段扩张段巨噬细胞数量明显减少，Cajal间质细胞数量有所恢复。接下来，我们采用含有TNF-a的培养基培养Cajal间质细胞，发现其表型受体c-kit表达明显下调。经生物信息学软件Targetscan预测，发现miR221是调控c-kit的关键因素，我们以斑马鱼作为模式动物，使用LPS喂养后发现TNF-α表达上调，c-kit表达明显下调，荧光示踪剂显示斑马鱼肠道传输速率明显下降，同时miR221高表达；构建吗啡啉修饰的反义寡核苷酸链抑制miR221表达后，再用LPS喂养发现c-kit基因及蛋白水平有恢复，并且肠道蠕动速率亦有恢复。这些研究证明了巨结肠肠道巨噬细胞活化对Cajal间质细胞的表型改变起重要作用，并从机理上证明了影响c-kit表达的关键靶点，这些研究为临床上逆转Cajal间质细胞表型，防止HD术后便秘复发奠定了理论基础。