【作者】 蔡育；

【导师】 赵怡芳；

【作者基本信息】 武汉大学 ， 口腔临床医学， 2010， 博士

【摘要】 婴幼儿血管瘤(Infantile Hemangiomas, IH,下文简称为血管瘤)是儿童最常见的肿瘤之一,60-80%的病变累及颌面部。它是一种非常特殊的肿瘤,包括血管生成和血管退化两种病理生理过程,根据其主要临床病理特点可分为快速增生期、退化期和退化完成期。最近有研究发现,增生期血管瘤内有大量的髓系细胞(myeloid cells),部分血管瘤内皮细胞共表达髓系细胞标志物,如CD14,CD45, CD32, CD15及CD83；我们的前期研究发现部分血管瘤内皮细胞共表达巨噬细胞标志物CD68；另有研究发现血管瘤组织中大量存在CD8阳性的细胞毒性T细胞,并且免疫调节剂2,3-吲哚胺二氧化酶(Indoleamine 2,3-dioxygenase, IDO)高表达于增生期血管瘤组织中,而在消退的过程中逐渐表达减少。所有这些研究都表明免疫及免疫介导的炎性过程在血管瘤的发生发展过程中可能起到重要的作用。新发现的生物活性肽——同种异体移植炎症因子1 (allograft inflammatory factor-1, AIF-1)不仅是巨噬细胞激活的标志物,更是免疫反应的重要调控因子。因此,我们推测AIF-1有在血管瘤发生发展中起到重要的调控作用,本研究旨在探讨AIF-1在血管瘤发生发展中的表达及其可能的作用机制。研究内容分为以下三部分：第一部分同种异体移植炎症因子1(AIF-1)在婴幼儿血管瘤中的表达及定位的研究目的：检测AIF-1在血管瘤组织及对照组织(包括脉管畸形、化脓性炎性肉芽肿、胎盘、舌鳞状细胞癌及正常皮肤)中的表达,以及对比AIF-1在血管瘤病理过程中各个时期的表达差异；方法：用免疫组织化学S-P法对19例血管瘤组织及对照组织(包括26例脉管畸形、24例化脓性炎性肉芽肿、3例胎盘、10例舌鳞状细胞癌及5例正常皮肤)的连续切片进行AIF-1及CD34染色,并对血管瘤组织进行AIF-1及CD34的免疫荧光双染；结果：19例血管瘤组织中有17例(89%)中可检测到AIF-1的表达,AIF-1表达于血管瘤内皮细胞的胞浆中,而24例化脓性炎性肉芽肿、26例静脉畸形、5例正常皮肤、10例舌癌及5例胎盘的血管中均未见AIF-1的表达；并且AIF-1在血管瘤组织三个时期均高表达,无显著性差异(P>0.05)；结论：AIF-1特异性地表达于血管瘤内皮细胞,而化脓性炎性肉芽肿、静脉畸形、正常皮肤的血管、胎盘及恶性肿瘤新生血管均不表达AIF-1,提示AIF-1可以作为血管瘤内皮细胞特殊的生物学标志物。第二部分同种异体移植炎症因子1(AIF-1)促进内皮细胞激活及其机制的研究目的：本部分研究旨在通过探讨AIF-1对内皮细胞的作用,来探讨AIF-1对血管瘤发生发展中可能存在的作用及其机制；方法：构建AIF-1的表达质粒pcDNA3.1(-)-AIF-1转染正常血管内皮细胞系HUV-EC-Cs,建立AIF-1过表达的内皮细胞株；通过MTT、流式细胞周期实验检测内皮细胞的增殖,内皮细胞划痕及迁移实验检测内皮细胞的迁移能力,成管实验及鸡胚尿囊膜成血管实验检测内皮细胞的分化能力、半定量的RT-PCR、Real time PCR及ELISA实验方法检测血管内皮生长因子(vascular endothelial growth factor, VEGF)、碱性成纤维细胞生长因子(basic fibroblast growth factor, b-FGF)、金属蛋白酶组织抑制剂1(tissue inhibitor of metalloproteinase 1, TIMP-1)、单核细胞趋化蛋白1 (monocyte chemoattractant protein-1, MCP-1)及粒细胞集落刺激因子(granulocyte colony-stimulating factor, G-CSF)的mRNA和蛋白水平；结果：正常内皮细胞不表达AIF-1,转染后AIF-1的表达显著增加；MTTT及流式细胞周期实验显示内皮细胞表达AIF-1后增殖能力显著增强(p<0.05),停留于静止期(G0／G1)的细胞比例明显减少,而处于DNA合成期(S)及分裂期(G2／M)的细胞比例明显增加；细胞划痕及迁移实验同样证实AIF-1能促进内皮细胞迁移；同时,成管实验及鸡胚尿囊膜成血管实验显示AIF-1能促进血管生成；并且,AIF-1可以上调内皮细胞bFGF的表达,而对VEGF、TIMP-1、MCP-1及G-CSF的表达无影响；结论：AIF-1有可能是通过上调bFGF的表达来促进内皮细胞增殖、迁移及血管生成,从而影响血管瘤的发生发展。第三部分在婴幼儿血管瘤中同种异体移植炎症因子1(AIF-1)上游调控因素的研究目的：探讨AIF-1在血管瘤中表达的原因；方法：用bFGF、VEGF、胰岛素样生长因子2 (insulin-like growth factor 2, IGF 2)及低氧刺激血管内皮细胞,作用24h或48h后,运用RT-PCR的方法检测AIF-1的表达；结果：bFGF、VEGF、IGF 2的浓度及低氧均不能诱导AIF-1的表达,并且不依赖于浓度及作用时间；结论：在血管瘤发生发展过程中起重要作用的生长因子并不影响AIF-1的表达。更多还原

【Abstract】 Infantile hemangiomas are benign tumors that exhibit an early and rapid proliferation phase within the first year of life, followed by a slower but steady involution phase that may last up to 5 to 10 years. Recent work has demonstrated the presence of large numbers of myeloid cells in proliferating hemangiomas and suggested that these cells might be involved in promoting growth. Ritter et al. reported that endothelial cells of hemangiomas coexpress some markers typically associated with myeloid cells, including CD14, CD45, CD32, CD15 and CD83, demonstrating a close relationship between myeloid and haemangioma endothelialcells. Furthermore, investigations into possible mechanisms of hemangioma regression suggest that this process is immune mediated. Infantile hemangiomas recruit a significant number of T cells, most of which are CD8-positive cytotoxic T cells. DNA microarray analysis has identified indoleamine 2,3-dioxygenase, an enzyme involved in the degradation of tryptophan that is critical to the function of T cells, as a potential modulator of the immune response in hemangiomas. Indoleamine 2,3-dioxygenase messenger RNA is present in high levels in proliferating hemangiomas and decreases significantly during involution, thus raising the possibility that inhibition of T cells could contribute to the slow regression of hemangiomas. All these evidences suggest that immune and immune-mediated inflammatory events may contribute to the pathogenesis of hemangiomas.The allograft inflammatory factor-1 (AIF-1) is a 17-kDa interferon (IFN)-y-inducible Ca2+-binding EF-hand protein that is encoded within the HLA class III genomic region on chromosome 6p21.3, which is known for clusters of genes involved in the inflammatory response. AIF-1 was originally cloned from activated macrophages in human and rat atherosclerotic allogenic heart grafts undergoing chronic transplant rejection. And resent research found that AIF-1 was not only a controller of various host responses to inflammatory stimuli, but also a modulator of immune responses. Therefore, we proposed that AIF-1 may play potential roles in the pathogenesis of hemangiomas. In the present study, we tried to explore the expression and possible effects of AIF-1 in the pathogenesis of hemangiomas. And there are three parts in this study.Part One: Expression of allograft inflammatory factor-1 in hemangiomas:its implication in the progression of hemangiomaObjective:To investigate the expression and location of AIF-1 in infantile hemangioma and compare the expression levels of AIF-1 among the three stages of hemangiomas. Methods:In a retrospective study of 19 hemangiomas in the oral and facial regions, we assessed lesional immunoreactivities for AIF-1. Negative controls were similarly confirmed, including 24 pyogenic granulomas,26 vascular malformations,5 samples of normal skin,10 squamous cell carcinomas of the tongue and 3 placentas. And the Double immunofluorescence labeling and series section was use to investigate the location of AIF-1 in hemangiomas and the control tissue.Results:Of all samples, intense immunoreactivity for AIF-1 was detected in 17 of 19 (89.47%) hemangioma specimens, with specific locations in endothelial cells. The intensity of AIF-1 immunostaining did not show remarkable difference among proliferating, involuting and involuted hemangiomas. However, no specific immunoreaction for AIF-1 was observed in vessels of vascular malformations, normal skin, squamous cell carcinomas, pyogenic granulomas or placental tissues.Conclusions:The exclusive expression of AIF-1 on endothelial cells of hemangiomas suggests that it possibly plays a significant role in the pathophysiology and progression of hemangioma. AIF-1 can be used as an additional biomarker for infantile hemangiomas.Part Two: The potential roles of allograft inflammatory factor-1 in the pathogenesis of hemangiomas:AIF-1 promotes the proliferation, migration and angiogenesis of human endothelial cells (HUV-EC-C) probably by up-regulation of bFGFObjective:To explore the impact of AIF-1 alterations on human endothelial cells (HUV-EC-C). Methods:Stable introduction of AIF-1 to human umbilical vein endothelial cell line (HUV-EC-C) in vitro was used to evaluate the role of AIF-1 by examining the change of cell proliferation, cell cycle, cell migration, angiogenesis and the expression level of proliferative and angiogenic related factors. Cell proliferation and cycle was measured by MTT assay and Flow Cytometry, Cell migration was assessed by the wound healing assay and the endothelial cell migration assay, and the ability of angiogenesis was investigated by the tuber formation assay and chicken chorioallantoic membrane assay. Furthermore, the expression levels of G-CSF, VEGF-a, bFGF MCP-1 and TIMP-1 was assessed by the semi-quantitative RT-PCR, Real time PCR and ELISA assay. Results:AIF-1 enhanced the proliferation,migration and angiogenesis of the endothelial cells and promoted G0/G1-to-S-phase transition, accompanied by up-regulation the expression of bFGF (P<0.05), while AIF-1 could not influence the expression of G-CSF, VEGF-a, MCP-1 and TIMP-1. Conclusions:Our studies demonstrated that AIF-1 could promote the proliferation, migration and angiogenesis of endothelial cells, probably through augmenting the expression of bFGF. The impact of AIF-1 on endothelial cells would stimulate angiogenesis, and consequently influence the progression of infantile hemangiomas.Part Three: Study on the up-stream regulator of allograft inflammatory factor-1 in infantile hemangiomasObjective:To investigate the possible up-stream regulator of AIF-1 in hemangiomas.Methods:The endothelial cells (HUV-EC-Cs) were treated by VEGF, bFGF, IGF-2 or hypoxia, respectively, for 24h or 48h. And then the mRNA of AIF-1 was assessed by RT-PCR. Results:Hypoxia, VEGF, bFGF and IGF-2, independent on the concentration and the time, could not induce the expression of AIF-1 in HUV-EC-C cells. Conclusions:Though AIF-1 is a cytokine-responsive transcription, the factors which play essential roles in the progression of infantile hemangiomas could not induce the expression of AIF-1. And further investigation is needed to determine which factor induces the expression of AIF-1 in IH.更多还原