【作者】 张凌；

【导师】 郑家伟；

【作者基本信息】 上海交通大学 ， 口腔临床医学， 2014， 博士

【摘要】 目的：利用婴幼儿血管瘤（IH）干细胞（HemSC）联合脐静脉内皮细胞（HUVEC）结合肌注雌激素,探索构建血管瘤裸鼠模型的新方法，研究雌激素（17β-雌二醇，E2）对血管瘤的作用机制，探讨Notch信号通路在血管瘤发生、发展过程中的调控作用。方法：使用CD133免疫磁珠分选方法，从增殖期IH组织中分选出HemSC，采用多向分化实验，证实其多向分化能力。对体外培养的HemSC分别给予10-9M、10-8M、10-7M、10-6M及10-5M浓度的E2作用48～72h，通过MTT实验、酶联免疫吸附试验(Elisa)、反转录PCR(RT-PCR)、实时荧光定量PCR(real-time PCR)和免疫印迹(Western blotting)，研究E2对HemSC的增殖以及对FGF2、VEGF-A、雌激素受体（estrogen receptor ERα）、Notch基因家族受体Notch1及配体Jagged1影响，来探讨E2的作用机制。将HemSC与脐静脉内皮细胞（HUVEC）联合注入BALB/c-nu裸鼠皮下，随机分为5组，每组4只，每周注射1次不同剂量（0、0.01mg/只、0.1mg/只、1mg/只）的17β-雌二醇（100μLDMSO溶解），不加药物组肌肉注射DMSO100μL/只作为对照组，不加药物也不注射DMSO组作为空白对照组，进行He染色，测定微血管密度(MVD)，并以免疫组织化学方法证实。结果：使用磁珠分选技术，可从IH组织中分选出HemSC，虽比例很低，但具有向脂肪细胞、成骨细胞和内皮细胞的多向分化能力。与HUVEC联合注射并结合肌注E2，能够形成更多的微血管。随着E2浓度的不断升高，MVD也逐渐增大，而且血管瘤内皮细胞向脂肪细胞转化的时间也相应延长。体外实验发现，当E2浓度为10-9M～10-6M时，能够明显促进HemSC增殖。高于10-6M后,HemSC的增殖变得不明显。E2能明显促进HemSC表达VEGF-A、FGF2mRNA和蛋白，基因与蛋白的表达与浓度梯度呈线性关系。免疫组织化学染色示，ERα及葡萄糖转运蛋白1（Glut-1）在IH组织中高表达。HemSC中，Notch基因家族Notch1及配体Jagged1，随E2浓度的增高而表达增强。结论：将HemSC和HUVEC混合注入BALB/c-nu裸鼠皮下，结合肌注10-6M E2，能够更好地构建血管瘤动物模型，增加MVD。E2能够有效促进HemSC增殖，并抑制其向脂肪细胞转化，在HemSC分化为血管内皮细胞、周细胞的过程中起到了重要的调控作用。ERα对Notch信号通路中受体Notch1及配体Jagged1具有调控作用，雌激素受体可能通过对Notch信号通路的调控，来影响HemSC分化为其他细胞的能力。雌激素促血管生成的作用主要通过上调VEGF-A、FGF2的表达，使两者协同而实现。这一结果将帮助我们更好地理解血管瘤发病的分子机制。更多还原

【Abstract】 Objective: The aim of the study was to establish mice model with hemangioma derivedstem cell (HemSC) and human umbilical vein endothelial cell(HUVEC),combined withestrogen（E2） injection. And investigate the mechanism of E2on proliferatinghemangioma.Methods: HemSCs were selected from proliferating hemangioma tissue by CD133taggedmagnetic-activated cell sorting technique. Their ability of multilineage differentiation wasverified by multiplex differentiation induction experiment. HemSCs were cultured with10-9M,10-8M,10-7M,10-6M and10-5M E2for48to72h respectively; then the impact onproliferation, mRNA and protein expression of FGF2, VEGF-A, ERα, Notch1andJagged1were investigated by MTT, RT-PCR, real-time PCR, Elisa and Western blotting,respectively. Finally, HemSC and HUVEC were mixed，then injected subcutaneously into20flank of BALB/c-nu mice that were randomly divided into5groups，group1(n=4) withgroup1(n=4) with0.01mg E2, group2(n=5) with0.1mg E2, group3(n=5) with1mg E2，group4(n=5) with0.1mg DMSO as control, group3(n=5) with0mg E2administeredintramuscularly every week. Two and4weeks later, the subcutaneous implants wereharvested and made into tissue spices for micro vessel density assay (MVD) andimmunohistochemistry.Results: Few HemSCs were isolated from IH tissue by MACS. HemSCs possessed theability of multilineage differentiation into adipocyte, osteoblast and endothelial cell. E2could increase the expression of FGF2, VEGF-A, ERα, Notch1and Jagged1in HemSCwith the optimal concentration from10-9M to10-5M. Two weeks after injection into BALB/c-nu mice with E2, HemSC and HUVEC formed micro vessels，the quantity ofblood vessel was greatly increased。Glucose transporter-1(Glut-1) which was a specificmarker of IH was positively stained. E2in normal concentration could increase theviability and proliferation of HemSC, and stimulation of angiogenesis.Conclusions: HemSCs possess the unique ability of robust proliferation and multilineagedifferentiation. HemSCs and HUVEC were co-injected into immunodeficient miceresuspended in Matrigel administered intramuscularly. E2modulation of Notch pathway ismediated by estrogen receptors. E2affects several Notch pathway components in HemSC,leading to an activation of the Notch pathway and to a modulation of VEGF-A whenNotch signalling is inhibited. These results contribute to our understanding of themolecular mechanisms of hemangioma by uncovering a novel role of E2in the Notchsignalling-mediated modulation of HemSC.更多还原