本文選題：巨噬細(xì)胞　切入點(diǎn)：血管瘤　出處：《武漢大學(xué)》2014年博士論文

【摘要】：第一部分巨噬細(xì)胞標(biāo)志物在血管瘤中的表達(dá)及分布 目的：巨噬細(xì)胞在腫瘤發(fā)生發(fā)展、動脈粥樣硬化及胰島素抵抗性肥胖癥等病理過程均發(fā)揮著重要作用,本部分研究目的在于探究巨噬細(xì)胞及其亞型M1及M2型巨噬細(xì)胞標(biāo)志物在血管瘤各期中的表達(dá)及分布,并分析其與細(xì)胞增殖、脂肪分化相關(guān)指標(biāo)及Akt、Erk:1/2信號通路活化之間的關(guān)系。 方法：利用免疫組織化學(xué)分別檢測巨噬細(xì)胞及其亞型標(biāo)志物CD68、HLA-DR及CD163,及細(xì)胞增殖抗原Ki67、脂肪分化調(diào)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達(dá)水平；采用免疫組織熒光檢測CD68+HLA-DR+M1及CD68+CD163+M2型巨噬細(xì)胞在血管瘤中的分布；通過聚類分析對上述指標(biāo)進(jìn)行相關(guān)性分析。采用實(shí)時(shí)定量PCR技術(shù)檢測巨噬細(xì)胞相關(guān)因子在血管瘤組織中的表達(dá)。 結(jié)果：多數(shù)血管瘤組織中(12/13)均可檢測到CD68、HLA-DR和CD163的表達(dá),同時(shí)CD68+HLA-DR+的M1型巨噬細(xì)胞及CD68+CD163+的M2型巨噬細(xì)胞在血管瘤增生期及消退期均可被發(fā)現(xiàn)；巨噬細(xì)胞,包括M1及M2型巨噬細(xì)胞,主要分布于血管瘤組織的間質(zhì)部分；同時(shí)定量分析顯示巨噬細(xì)胞標(biāo)志物在增生期血管瘤表達(dá)明顯高于消退期,具有統(tǒng)計(jì)學(xué)差異。聚類分析顯示巨噬細(xì)胞標(biāo)志物與VEGF、Ki67、磷酸化Akt呈正相關(guān),與PPARy呈負(fù)相關(guān)。實(shí)時(shí)定量PCR結(jié)果顯示巨噬細(xì)胞相關(guān)因子TNF-α, IL-1β及TGF-β在血管瘤增生期內(nèi)高表達(dá)。 結(jié)論：多數(shù)血管瘤組織可檢測到巨噬細(xì)胞及其亞型的分布,主要位于血管瘤組織的間質(zhì)部分。巨噬細(xì)胞,包括Ml及M2型巨噬細(xì)胞在增生期的數(shù)量明顯高于消退期。巨噬細(xì)胞標(biāo)志物CD68、HLA-DR及CD163與VEGF、Ki67、磷酸化Akt的表達(dá)呈正相關(guān),與PPARγ呈負(fù)相關(guān),提示巨噬細(xì)胞可能參與了血管瘤的發(fā)展及消退。 第二部分血管瘤干細(xì)胞分離、鑒定及血管瘤裸鼠模型的建立 目的：血管瘤干細(xì)胞(hemangiomastem cell, HemSC)是利用CD133抗體標(biāo)記從增生期血管瘤組織中分離的具有增殖、克隆形成及多項(xiàng)分化潛能的一類干細(xì)胞,將其注射于裸鼠皮下可模擬人類血管瘤的發(fā)展及消退,成為研究血管瘤發(fā)病機(jī)制的有力工具。本部分研究目的在于利用CD133抗體磁珠篩選出血管瘤干細(xì)胞,鑒定其表征后嘗試建立血管瘤裸鼠模型。 方法：利用CD133抗體磁珠結(jié)合磁性細(xì)胞篩選系統(tǒng)分離增生期血管瘤組織中的血管瘤干細(xì)胞,采用流式細(xì)胞術(shù)對其表面標(biāo)志物進(jìn)行分析,進(jìn)一步利用MTT增殖試驗(yàn)、克隆形成試驗(yàn)、分化試驗(yàn)檢測分離出的血管瘤干細(xì)胞的細(xì)胞學(xué)特點(diǎn)。將分離的血管瘤干細(xì)胞與基質(zhì)膠混合后注射于裸鼠皮下建立血管瘤裸鼠模型。 結(jié)果：利用CD133抗體篩選的血管瘤干細(xì)胞呈現(xiàn)出成纖維細(xì)胞樣形態(tài),具有較強(qiáng)的增殖能力及克隆形成能力,同時(shí)表達(dá)干細(xì)胞轉(zhuǎn)錄因子AML1及Oct-4；分離的血管瘤干細(xì)胞膜表面內(nèi)皮細(xì)胞標(biāo)志分子CD31、CD34、CD146表達(dá)呈弱陽性,間質(zhì)細(xì)胞標(biāo)志物CD105、CD90呈陽性,間質(zhì)干細(xì)胞標(biāo)志物STRO-1表達(dá)水平較低；分化試驗(yàn)顯示其具有多向分化潛能,可向脂肪細(xì)胞、成骨細(xì)胞及內(nèi)皮細(xì)胞分化。利用血管瘤干細(xì)胞建立的血管瘤裸鼠模型具有血管形成、脂肪分化等特點(diǎn),可部分模擬人類血管瘤發(fā)展特點(diǎn)。 結(jié)論：分離培養(yǎng)的血管瘤干細(xì)胞具有快速增殖、克隆形成及多向分化等能力,表達(dá)間質(zhì)細(xì)胞及內(nèi)皮細(xì)胞標(biāo)志物。利用其可建立具有人類血管瘤發(fā)展特點(diǎn)的血管瘤裸鼠模型。 第三部分巨噬細(xì)胞對血管瘤干細(xì)胞增殖、分化的影響及機(jī)制研究 目的：本部分研究目的在于通過建立血管瘤干細(xì)胞與巨噬細(xì)胞的共培養(yǎng)體系,在體外環(huán)境中檢測不同極性巨噬細(xì)胞(M1及M2型巨噬細(xì)胞)對血管瘤干細(xì)胞增殖及脂肪分化的調(diào)控作用及機(jī)制,同時(shí)檢測血管瘤干細(xì)胞對單核細(xì)胞的影響。 方法：利用條件培養(yǎng)基及Transwell系統(tǒng)建立血管瘤干細(xì)胞與單核/巨噬細(xì)胞之間的間接共培養(yǎng)體系,采用EdU摻入試驗(yàn)、MMT試驗(yàn)檢測巨噬細(xì)胞對血管瘤干細(xì)胞增殖能力的影響,采用油紅O染色檢測巨噬細(xì)胞對血管瘤干細(xì)胞脂肪分化的作用,并利用蛋白芯片、免疫印跡等技術(shù)探究巨噬細(xì)胞對血管瘤干細(xì)胞產(chǎn)生作用的分子機(jī)制,利用信號通路特異性抑制劑驗(yàn)證相關(guān)信號通路的作用。 結(jié)果：M1及M2型巨噬細(xì)胞條件培養(yǎng)基均可促進(jìn)血管瘤干細(xì)胞的增殖、抑制其脂肪分化,采用胞內(nèi)信號蛋白芯片及免疫印跡檢測顯示巨噬細(xì)胞條件培養(yǎng)基可活化血管瘤干細(xì)胞內(nèi)Akt及Erk1/2信號通路,利用小分子特異性抑制劑LY294002及PD98059可明確Akt的活化介導(dǎo)了巨噬細(xì)胞促進(jìn)血管瘤干細(xì)胞的增殖,Erk1/2的活化介導(dǎo)了巨噬細(xì)胞抑制血管瘤干細(xì)胞的脂肪分化。利用Transwell間接共培養(yǎng)系統(tǒng)發(fā)現(xiàn)HemSC可促進(jìn)THP-1的增殖及Akt的活化,對THP-1的分化無明顯影響。 結(jié)論：巨噬細(xì)胞可通過活化血管瘤干細(xì)胞內(nèi)的Akt及Erkl/2信號通路促進(jìn)其增殖并抑制其脂肪分化,血管瘤干細(xì)胞則可促進(jìn)單核細(xì)胞系THP-1的增殖,對其分化無明顯影響。 第四部分巨噬細(xì)胞在血管瘤裸鼠模型中的作用研究 目的：上述研究顯示巨噬細(xì)胞可能在血管瘤的發(fā)展過程中起到重要作用,本部分研究目的在于建立巨噬細(xì)胞參與的血管瘤裸鼠模型,并基于此模型探究巨噬細(xì)胞在血管瘤增生期及消退期的作用及機(jī)制,驗(yàn)證上述實(shí)驗(yàn)部分的結(jié)果。 方法：通過按一定比例混合血管瘤干細(xì)胞及單核細(xì)胞系THP-1(HemSC:THP-1=4:1),注射于裸鼠背部皮下,分別于7天、14天、28天及56天后收獲血管瘤裸鼠模型標(biāo)本,利用免疫組織化學(xué)、免疫組織熒光、實(shí)時(shí)定量PCR等技術(shù)檢測巨噬細(xì)胞標(biāo)志物及相關(guān)因子的表達(dá),及細(xì)胞增殖、脂肪分化相關(guān)指標(biāo)的表達(dá)及Akt、Erk1/2信號通路的活化。 結(jié)果：混合THP-1的血管瘤模型(THP-1組)相對于單純注射HemSC模型(對照組),標(biāo)本組織具有更高的細(xì)胞密度,其中cyclinD1染色強(qiáng)度明顯增高,提示巨噬細(xì)胞在血管瘤模型中促進(jìn)了血管瘤細(xì)胞的增殖。同時(shí),THP-1組標(biāo)本具有更高的微血管密度(MVD)及CD31表達(dá),提示巨噬細(xì)胞促進(jìn)了血管瘤中的血管形成。THP-1組標(biāo)本的脂質(zhì)空泡減少及PPARγ表達(dá)明顯減少,提示巨噬細(xì)胞抑制了血管瘤的消退,同時(shí)發(fā)現(xiàn)巨噬細(xì)胞可促進(jìn)血管瘤細(xì)胞Akt及Erk1/2信號通路的活化。 結(jié)論：利用巨噬細(xì)胞參與的血管瘤裸鼠模型,證實(shí)巨噬細(xì)胞可促進(jìn)血管瘤的增殖及血管形成、抑制其脂肪分化,活化血管瘤細(xì)胞的Akt及Erkl/2信號通路,提示巨噬細(xì)胞可能在促進(jìn)血管瘤增生期的進(jìn)展,抑制其消退方面發(fā)揮重要作用,且這種作用可能依賴于Akt及Erkl/2信號通路。
[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.

【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R732.2

【共引文獻(xiàn)】

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