巨噬细胞在婴幼儿血管瘤发展过程中的作用

发布时间：2018-04-08 20:34

本文选题：巨噬细胞　切入点：血管瘤　出处：《武汉大学》2014年博士论文

【摘要】：第一部分巨噬细胞标志物在血管瘤中的表达及分布目的：巨噬细胞在肿瘤发生发展、动脉粥样硬化及胰岛素抵抗性肥胖症等病理过程均发挥着重要作用,本部分研究目的在于探究巨噬细胞及其亚型M1及M2型巨噬细胞标志物在血管瘤各期中的表达及分布,并分析其与细胞增殖、脂肪分化相关指标及Akt、Erk:1/2信号通路活化之间的关系。方法：利用免疫组织化学分别检测巨噬细胞及其亚型标志物CD68、HLA-DR及CD163,及细胞增殖抗原Ki67、脂肪分化调控分子PPARγ、磷酸化Akt及Erkl/2在血管瘤增生期及消退期中的表达水平；采用免疫组织荧光检测CD68+HLA-DR+M1及CD68+CD163+M2型巨噬细胞在血管瘤中的分布；通过聚类分析对上述指标进行相关性分析。采用实时定量PCR技术检测巨噬细胞相关因子在血管瘤组织中的表达。结果：多数血管瘤组织中(12/13)均可检测到CD68、HLA-DR和CD163的表达,同时CD68+HLA-DR+的M1型巨噬细胞及CD68+CD163+的M2型巨噬细胞在血管瘤增生期及消退期均可被发现；巨噬细胞,包括M1及M2型巨噬细胞,主要分布于血管瘤组织的间质部分；同时定量分析显示巨噬细胞标志物在增生期血管瘤表达明显高于消退期,具有统计学差异。聚类分析显示巨噬细胞标志物与VEGF、Ki67、磷酸化Akt呈正相关,与PPARy呈负相关。实时定量PCR结果显示巨噬细胞相关因子TNF-α, IL-1β及TGF-β在血管瘤增生期内高表达。结论：多数血管瘤组织可检测到巨噬细胞及其亚型的分布,主要位于血管瘤组织的间质部分。巨噬细胞,包括Ml及M2型巨噬细胞在增生期的数量明显高于消退期。巨噬细胞标志物CD68、HLA-DR及CD163与VEGF、Ki67、磷酸化Akt的表达呈正相关,与PPARγ呈负相关,提示巨噬细胞可能参与了血管瘤的发展及消退。第二部分血管瘤干细胞分离、鉴定及血管瘤裸鼠模型的建立目的：血管瘤干细胞(hemangiomastem cell, HemSC)是利用CD133抗体标记从增生期血管瘤组织中分离的具有增殖、克隆形成及多项分化潜能的一类干细胞,将其注射于裸鼠皮下可模拟人类血管瘤的发展及消退,成为研究血管瘤发病机制的有力工具。本部分研究目的在于利用CD133抗体磁珠筛选出血管瘤干细胞,鉴定其表征后尝试建立血管瘤裸鼠模型。方法：利用CD133抗体磁珠结合磁性细胞筛选系统分离增生期血管瘤组织中的血管瘤干细胞,采用流式细胞术对其表面标志物进行分析,进一步利用MTT增殖试验、克隆形成试验、分化试验检测分离出的血管瘤干细胞的细胞学特点。将分离的血管瘤干细胞与基质胶混合后注射于裸鼠皮下建立血管瘤裸鼠模型。结果：利用CD133抗体筛选的血管瘤干细胞呈现出成纤维细胞样形态,具有较强的增殖能力及克隆形成能力,同时表达干细胞转录因子AML1及Oct-4；分离的血管瘤干细胞膜表面内皮细胞标志分子CD31、CD34、CD146表达呈弱阳性,间质细胞标志物CD105、CD90呈阳性,间质干细胞标志物STRO-1表达水平较低；分化试验显示其具有多向分化潜能,可向脂肪细胞、成骨细胞及内皮细胞分化。利用血管瘤干细胞建立的血管瘤裸鼠模型具有血管形成、脂肪分化等特点,可部分模拟人类血管瘤发展特点。结论：分离培养的血管瘤干细胞具有快速增殖、克隆形成及多向分化等能力,表达间质细胞及内皮细胞标志物。利用其可建立具有人类血管瘤发展特点的血管瘤裸鼠模型。第三部分巨噬细胞对血管瘤干细胞增殖、分化的影响及机制研究目的：本部分研究目的在于通过建立血管瘤干细胞与巨噬细胞的共培养体系,在体外环境中检测不同极性巨噬细胞(M1及M2型巨噬细胞)对血管瘤干细胞增殖及脂肪分化的调控作用及机制,同时检测血管瘤干细胞对单核细胞的影响。方法：利用条件培养基及Transwell系统建立血管瘤干细胞与单核/巨噬细胞之间的间接共培养体系,采用EdU掺入试验、MMT试验检测巨噬细胞对血管瘤干细胞增殖能力的影响,采用油红O染色检测巨噬细胞对血管瘤干细胞脂肪分化的作用,并利用蛋白芯片、免疫印迹等技术探究巨噬细胞对血管瘤干细胞产生作用的分子机制,利用信号通路特异性抑制剂验证相关信号通路的作用。结果：M1及M2型巨噬细胞条件培养基均可促进血管瘤干细胞的增殖、抑制其脂肪分化,采用胞内信号蛋白芯片及免疫印迹检测显示巨噬细胞条件培养基可活化血管瘤干细胞内Akt及Erk1/2信号通路,利用小分子特异性抑制剂LY294002及PD98059可明确Akt的活化介导了巨噬细胞促进血管瘤干细胞的增殖,Erk1/2的活化介导了巨噬细胞抑制血管瘤干细胞的脂肪分化。利用Transwell间接共培养系统发现HemSC可促进THP-1的增殖及Akt的活化,对THP-1的分化无明显影响。结论：巨噬细胞可通过活化血管瘤干细胞内的Akt及Erkl/2信号通路促进其增殖并抑制其脂肪分化,血管瘤干细胞则可促进单核细胞系THP-1的增殖,对其分化无明显影响。第四部分巨噬细胞在血管瘤裸鼠模型中的作用研究目的：上述研究显示巨噬细胞可能在血管瘤的发展过程中起到重要作用,本部分研究目的在于建立巨噬细胞参与的血管瘤裸鼠模型,并基于此模型探究巨噬细胞在血管瘤增生期及消退期的作用及机制,验证上述实验部分的结果。方法：通过按一定比例混合血管瘤干细胞及单核细胞系THP-1(HemSC:THP-1=4:1),注射于裸鼠背部皮下,分别于7天、14天、28天及56天后收获血管瘤裸鼠模型标本,利用免疫组织化学、免疫组织荧光、实时定量PCR等技术检测巨噬细胞标志物及相关因子的表达,及细胞增殖、脂肪分化相关指标的表达及Akt、Erk1/2信号通路的活化。结果：混合THP-1的血管瘤模型(THP-1组)相对于单纯注射HemSC模型(对照组),标本组织具有更高的细胞密度,其中cyclinD1染色强度明显增高,提示巨噬细胞在血管瘤模型中促进了血管瘤细胞的增殖。同时,THP-1组标本具有更高的微血管密度(MVD)及CD31表达,提示巨噬细胞促进了血管瘤中的血管形成。THP-1组标本的脂质空泡减少及PPARγ表达明显减少,提示巨噬细胞抑制了血管瘤的消退,同时发现巨噬细胞可促进血管瘤细胞Akt及Erk1/2信号通路的活化。结论：利用巨噬细胞参与的血管瘤裸鼠模型,证实巨噬细胞可促进血管瘤的增殖及血管形成、抑制其脂肪分化,活化血管瘤细胞的Akt及Erkl/2信号通路,提示巨噬细胞可能在促进血管瘤增生期的进展,抑制其消退方面发挥重要作用,且这种作用可能依赖于Akt及Erkl/2信号通路。
[Abstract]:The expression and distribution of the first part of macrophage markers in hemangioma
Objective: macrophage in the occurrence and development of cancer, atherosclerosis and insulin resistance in obesity and other pathological processes play important roles, the purpose of this part is to explore the study of macrophages and its subtypes M1 and M2 type macrophage markers in the expression and distribution of hemangioma in different phases, and its correlations with cell proliferation, differentiation and related indexes of fat Akt. The relationship between the activation of Erk:1/2 signaling pathway.
Methods: to detect macrophage subtypes using immunohistochemical markers CD68, HLA-DR and CD163, and the proliferation of adipose differentiation antigen Ki67, molecular PPAR gamma, phosphorylation of Akt and Erkl/2 in proliferating hemangioma and the expression level in the period of extinction; by using immuno fluorescence detection of CD68+HLA-DR+M1 and CD68+CD163+M2 distribution of fabric in macrophages hemangioma; through cluster analysis of the above indicators for correlation analysis. The expression was detected by quantitative real-time PCR of macrophage related factors in hemangioma.
Results: the majority of hemangiomas (12/13) were detected in CD68, the expression of HLA-DR and CD163, and M1 CD68+CD163+ of the CD68+HLA-DR+ type macrophages and M2 macrophages in proliferating hemangioma and paracmasis can be found; macrophages, including M1 and M2 type macrophages, interstitial part mainly distributed in blood vessels the tumor tissue; while the quantitative analysis showed that macrophage marker expression in proliferative hemangioma was significantly higher than that in a recession, with statistical difference. Cluster analysis showed that macrophage markers and VEGF, Ki67, phosphorylation of Akt was positively correlated, negatively correlated with PPARy. Real time quantitative PCR results showed that macrophage associated factor TNF- alpha, IL-1 beta and TGF- beta high expression in proliferating hemangioma.
Conclusion: the distribution of most hemangiomas can be detected in macrophages and its subtypes, interstitial part mainly located in hemangioma tissues. Macrophages, including Ml and M2 macrophages in the number of proliferating macrophages was significantly higher than that of subsided period. Markers CD68, HLA-DR and CD163 and VEGF, Ki67, phosphorylated Akt expression was positively correlated and was negatively correlated with PPAR gamma, suggesting that macrophages may be involved in the development of hemangioma and subside.
Isolation, identification of second parts of hemangioma stem cells and the establishment of a nude mouse model of hemangioma
Objective: vascular tumor stem cells (hemangiomastem cell HemSC) is isolated from proliferation hemangioma by using CD133 antibody labeled with a kind of stem cell proliferation, clone formation and differentiation potential, it can be injected subcutaneously in nude mice and human hemangioma regression simulation development, has become a powerful tool for studies on pathogenesis of vascular the purpose of this part of the study. The tumor is using CD133 antibody beads screened hemangioma stem cells, identification of its attempt to establish a representation of hemangioma in nude mice.
Methods: Hemangioma by CD133 antibody combined with magnetic bead cell screening system separating proliferative hemangioma tissue stem cells, flow cytometry was used to analyze the surface marker, further use of MTT proliferation assay, colony formation assay, differentiation assay of hemangioma isolated stem cells. The cytological characteristics of hemangioma the separation of stem cells and Matrigel injection subcutaneously to establish Vascular Xenograft model.
Results: the hemangioma CD133 antibody screening cells showed a fibroblast like morphology, proliferation and cloning have strong ability to form at the same time, the expression of stem cell transcription factor AML1 and Oct-4; the surface of the endothelial cell membrane cell marker CD31, hemangioma of the isolated stem CD34, CD146 expression was weakly positive, interstitial cells markers CD105, CD90 positive mesenchymal stem cell marker STRO-1 expression level is low; the differentiation test showed that the multilineage differentiation potential into adipocytes, osteoblasts, and endothelial cell differentiation. The hemangioma vascular stem cells in nude mice were established with angiogenesis, adipose differentiation and other characteristics, can part of the simulation development characteristics of human hemangioma.
Conclusion: the isolated and cultured hemangioma stem cells have the ability of rapid proliferation, clone formation and multi differentiation, and express interstitial cells and endothelial cell markers.
The effect of third parts of macrophage on the proliferation and differentiation of hemangioma stem cells and its mechanism
Objective: the aim of this part of research is through the co culture system establishment of vascular tumor stem cells and macrophages in vitro, detection of different polarity of macrophages (M1 and M2 macrophages) on vascular tumor stem cell proliferation and adipogenic differentiation regulation, simultaneous detection of vascular tumor stem cells in mononuclear cells.
Methods: to establish vascular tumor stem cells and monocytes / macrophages between the indirect co culture system and Transwell based system using condition, using EdU incorporation test, MMT test of macrophage stem cells on the proliferation of hemangioma, using oil red O staining, macrophage stem cells differentiation into adipocytes of hemangioma and, using protein chip, molecular mechanism and immunoblotting of macrophages to produce effect on vascular tumor stem cells, to verify the relevant signaling pathway by specific inhibitors.
Results: M1 and M2 type macrophage conditioned medium can promote cell proliferation of vascular tumor stem, inhibit adipocyte differentiation, using signal protein chip and Western blot detection showed intracellular macrophage conditioned medium activation of vascular tumor stem Akt and Erk1/2 signaling pathways in cells, the use of small molecule inhibitors LY294002 and PD98059 can clear Akt activation mediated by macrophages promote cell proliferation of vascular tumor stem, the activation of Erk1/2 mediated inhibition of stem cell differentiation of macrophage fatty hemangioma. Indirect co culture system that HemSC can promote the proliferation and activation of Akt THP-1 by Transwell, had no significant effect on the differentiation of THP-1.
Conclusion: macrophages can promote proliferation and inhibit fat differentiation by activating Akt and Erkl/2 signaling pathways in hemangioma stem cells. Hemangioma stem cells can promote the proliferation of monocyte THP-1 and have no obvious effect on differentiation.
The role of fourth part of macrophage in the nude mouse model of hemangioma
Objective: the study shows that play an important role in the development process of macrophages may hemangioma, the purpose of this part of the study is to establish the Vascular Xenograft Model in macrophages, and based on this model of macrophages in proliferating hemangioma and regression effect and mechanism of the verification results of the experiment.
Methods: the mixed hemangioma according to a certain proportion of stem cells and mononuclear cell lines (HemSC:THP-1=4:1, THP-1) were injected subcutaneously in nude mice, respectively, in 7 days, 14 days, 28 days and 56 days after the harvest Vascular Xenograft model specimens by immunohistochemistry, immunofluorescence, real-time quantitative PCR technique to detect macrophage marker the expression and related factors, and cell proliferation, Akt expression and related indexes of adipose differentiation, activation of Erk1/2 signaling pathway.
Results: hemangioma model of hybrid THP-1 (THP-1 group) compared with injection of HemSC model (control group), the tissue has a higher cell density, the intensity of cyclinD1 staining increased significantly, suggesting that macrophages promoted the proliferation of hemangioma cells in hemangioma model. At the same time, THP-1 group of specimens with microvessel density and more high (MVD) and the expression of CD31, suggesting that lipid vacuoles in macrophages promote vascular tumor angiogenesis in the.THP-1 group were decreased and PPAR expression decreased, suggesting that macrophages inhibit hemangioma retrogression, also found that macrophages can promote the activation of vascular tumor cells Akt and Erk1/2 signaling pathway.
Conclusion: the Vascular Xenograft Model in macrophages, confirmed that macrophages can promote the formation of proliferation and vascular hemangioma, inhibit adipocyte differentiation, Akt and activation of Erkl/2 pathway in vascular tumor cells, suggesting that macrophages may promote the progression in proliferating hemangioma, plays an important role in inhibiting the subsided, and this effect may be depending on the Akt and Erkl/2 signaling pathway.

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R732.2

【共引文献】