beta肾上腺素受体在婴幼儿血管瘤发病中的作用研究
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摘要
背景
婴幼儿血管瘤(infantile hemangioma, IH)是多发于婴幼儿的一种良性肿瘤,出生后1个月到1岁内发病率高达8.7%-12.7%[1-3],影响患儿的外貌,部分严重者会涉及功能性器官,甚至危及生命。目前对该病的治疗主要采取手术、药物、激光等措施,或采用联合治疗的方案,但山于缺少对血管瘤病因和病理机制的认识,治疗效果不能令人满意[4]。
血管瘤的发病具有以下5个临床特点：①出生后即发病,1个月到1岁内发展迅速；1岁以后病变稳定并开始自发性消退,有着明显的迅速增殖期和自发性消退期的表现。②患者以女性居多,男女比率约为1：3-5[5-6]。③头颈部血管瘤约占全身发病的60%[7]。④白人罹患率是黑种人或黄种人的10-12倍[8]。⑤适当的激素治疗对该病有效[9-10]。
研究血管瘤发病机制的首要问题是建立符合IH病变自然进程的动物模型。目前报道过的IH动物模型建立方法主要有5种：①采用鸡冠和肉垂等富含血窦组织制备的皮肤毛细血管瘤模型[11]；②以化学药物如1,2-二甲肼(1,2-dimethylhydrazine dihydrochloride,1,2-DMH)诱发的恶性内皮细胞瘤或血管肉瘤[12]；③通过转基因方法将多瘤病毒(polyomavirus, Py)的中等T抗原(middle T antigen, MT)基因整合到小鼠正常基因序列的恶性多瘤病毒模型[13]；④将体外培养的血管瘤内皮细胞(hemangioma endothelial cell, HemEC)注射入裸鼠(免疫缺陷小鼠)皮下形成血管瘤[14]；⑤裸鼠皮下植入新鲜增殖期血管瘤组织[15]。
本课题针对以上模型中报道最多的HemEC皮下注射裸鼠模型和增殖期血管瘤组织块皮下移植裸鼠模型进行了比较,以选择最稳定、可靠的血管瘤动物模型,为接下来血管瘤发病机制的研究打下基础。
目的
1.探讨体外培养HemEC的可行性。
2.比较HemEC注射法和血管瘤组织块植入法裸鼠模型与血管瘤自然进程的相似性以及在血管瘤动物模型建立中的稳定性。
方法
1.收集山东大学齐鲁医院口腔颌面外科和临沂市肿瘤医院血管瘤科的血管瘤患者组织标本,取样条件为年龄小于3个月并且未接受任何治疗的2例手术切除病例。本研究已告知患儿监护人并签署知情同意书。
2.冲洗新鲜婴幼儿血管瘤组织块并修剪成1cm3大小,50%用于HemEC培养,50%用于构建组织块植入法裸鼠皮下血管瘤模型；另取少量组织固定,石蜡包埋后,免疫组织化学法(immunohistochemistry, IHC)鉴定葡萄糖转运蛋白1(glucose transporter1,GLUT1)的表达。
3.HemEC体外培养及鉴定：利用组织块贴壁培养法分离并培养HemEC;免疫细胞化学法(immunocytochemistry, ICC)检测内皮细胞标记物第八因子相关抗原因子(factor Ⅷ-related antigen, von Willebrand factor, vWF)和CD34在HemEC上的表达。
4.实验用裸鼠(BALB/c nude mice)24只,雌雄不限,鼠龄(28+3.2)d,体重(20.5+2.1)g,购白北京华阜康生物科技股份公司。24只裸鼠随机分为2组：组织块组和细胞注射组,每组分别12只。组织块组：双侧腋窝皮下植入处理过的1cm3大小组织块；细胞组：双侧腋窝皮下注射第3代HemEC。分别记录第7、30、60d两组血管瘤模型移植处瘤体的变化,包括瘤体尺寸、色泽和质地；在各时间点各组随机选取4只裸鼠切取移植处瘤体组织,固定,石蜡包埋后,苏木素-伊红(hematoxylin-eosin staining, HE)染色,镜下观察其组织学特点。
结果
1.IHC法鉴定GLUT1的表达检测血管瘤特异性标记物GLUT1在手术取得组织中的表达为强阳性,证明为增殖期血管瘤组织：HemEC皮下注射法血管瘤裸鼠模型第30d时,瘤体组织GLUT1呈阳性表达。
2.HemEC的培养及鉴定HemEC在组织块培养法下生长并传代,细胞贴壁生长,形状不规则,多为多角形或梭形,聚集生长；ICC法检测内皮细胞标记物vWF和CD34,均呈阳性表达。
3.HemEC皮下注射法血管瘤裸鼠模型的建立在12只裸鼠双侧腋下部皮下组织注射HemEC后,双侧全部成瘤；第7d时,瘤体约1mm×1mm×1mm大小,镜下见大量HemEC,并逐渐开始形成血管；第30d时,瘤体生长至5mm×4mm×3mm大小,镜下见大量密集生长的HemEC,并有大量HemEC围成的小血管,局部可见大血管；第60d时,瘤体为2mm×3mm×2mm大小,部分HemEC开始逐渐凋亡,血管萎缩或破裂。
4.增殖划血管瘤组织块移植法血管瘤裸鼠模型的建立移植组织块到裸鼠双侧腋下部皮下后,1周之内便有1只裸鼠因感染死亡,2只裸鼠因缝线被撕咬或挠抓而过早脱落,导致组织块从体内排出,其余9只裸鼠均双侧腋下成瘤。第7d时,移植块已与机体融合,镜下见HemEC围绕的密集小血管；第30d时,瘤体约2mm×2mm×2mm,镜下见大量移植细胞的变性、坏死,炎细胞遍布,仅有少量血管残留；第60d时,移植组织已完全融于机体,镜下可见移植物已脂肪化,仅见少量血管周围有类似残留移植物存在。
结论
1.血管瘤组织块贴壁法可以培养出HemEC。
2.组织块移植法血管瘤裸鼠模型因高失败率和无法大量建模的缺点,不能为本课题所用。
3.皮下注射人血管瘤内皮细胞的血管瘤裸鼠模型建模方法因其稳定性高,特异性强和可重复性的优点,是建立血管瘤的动物模型较为较理想的选择。
婴幼儿血管瘤(infantile hemangiomas, IH)是最常见的婴幼儿良性肿瘤之一,尽管对其组织学特点和发病机制的研究众多,但是一直没有将这些问题解释清楚。过去的几十年中,大量科研工作者致力于对该疾病发病机制的研究,也取得了一些进展,并建立了一些相应的学说,如胎盘绒毛膜学说,内皮细胞基因突变,以及外源性因子形成促进细胞生长的微环境等。但是仍然没有一种学说能够完全解释IH所有的临床和生理特征,因此需要一些突破性的发现来帮助我们理解并研究IH的发病机制,从而能够研发出对IH特异性更强的治疗方案。
口服普萘洛尔(一种非选择性beta1肾上腺素受体抑制剂)被报道能够有效治疗血管瘤,这一新发现使众多研究者将目光转移到了beta1肾上腺素受体(βadrenergic receptor, β-AR)与血管瘤之间的关系上。血管瘤由以血管瘤内皮细胞(hemangioma endothelial cell, HemEC)为主的多种细胞组成,p2-AR可在内皮细胞表面表达[1],而在HemEC上也发现了有β2和β3-AR的表达[2]。因此我们假设,普萘洛尔发挥治疗血管瘤的作用机制可能是通过以下3个方面实现的：①早期血管收缩。自主神经系统和肾上腺素在皿管管腔收缩与舒张的控制上起着至关重要的作用,肾上腺素依靠激活a1和β2-AR能够控制血管的收缩和舒张,所以单独的p2-AR抑制剂(如普萘洛尔)则可以阻断肾上腺素激活β2-AR引起的血管舒张,并且避免α1-AR被抑制,保留了对血管收缩的刺激。②中期抑制血管生成。在血管瘤增殖发育的旺盛阶段,有两个促血管生成因子的表达异常增高：血管内皮细胞生长因子(vascular endothelial growth factor, VEGF)和成纤维细胞生长因子(basic fibroblast growth factor, bFGF),在β2-AR抑制剂的阻断作用下,减少的VEGF分泌可有效的抑制血管生成[3]。③长期诱导HemEC的凋亡。HemEC的凋亡被多条信号通路所介导,其中β2-AR被抑制而诱发的下游信号通路所介导的细胞凋亡率最高[4]。
验证以上假说的关键是确定β2-AR在血管瘤发病机制中的重要作用,包括β2-AR的表达以及β2-AR表达被干扰后血管瘤症状的改变。以β2-AR为始点,对其下游信号通路的研究将是我们今后工作的重点。
目的
1.检测p-AR各亚型在血管瘤以及血管瘤内皮细胞中的表达情况。
2.观察β-AR各亚型被分别抑制后血管瘤发病情况的改变。
3.利用病毒转染携带干扰基因,探讨p2-AR在体内和体外血管瘤发病机制中的作用。
方法
1.获取新鲜增殖气期血管瘤、静脉畸形(vein malformation, VM)和淋巴管畸形(lymphatic malformation, LM)组织,固定包埋,制作切片,免疫组化法(immunohistochemistry, IHC)检测β1、β2、β3-AR的表达。
2.获取新鲜增殖期和消退期血管瘤组织,提取RNA和蛋白,qPCR和WB法检测β1、β2、p3-AR的表达。
3.获取新鲜增殖期血管瘤及其周围正常组织,提取RNA和蛋白,qPCR和WB法检测β1、β2、β3-AR的表达。
4.组织块法培养HemEC,以脐静脉内皮细胞(HUVEC)为对照,提取RNA和蛋白,qPCR和WB法检测β1、 β2、β3-AR的表达。
5.在体外,以梯度浓度普萘洛尔治疗HemEC, MTT法检测细胞增殖能力,成管实验检测细胞成管能力,经对比得到普萘洛尔治疗HemEC的最佳浓度。
6.在体外,用最佳浓度的p-AR各亚型特异性抑制剂治疗HemEC, MTT法检测细胞增殖能力,成管实验检测细胞成管能力,得到与普萘洛尔作用最接近的抑制剂所属的p-AR亚型。
7.设计并构建特异性干扰p2-AR慢病毒载体,转染HemEC,并提取RNA和蛋白,qPCR和WB法检测β2-AR的表达,以确定其病毒转染率。
8.在体外,用普萘洛尔和沉默β2-AR慢病毒(ADRB2-si)处理HemEC,再用高表达β2-AR慢病毒(ADRB2-hi)恢复甚至增加β2-AR的表达,MTT法检测细胞增殖能力,成管实验检测细胞成管能力的变化。
9.在HemEC皮下注射血管瘤模型上,用普萘洛尔和ADRB2-si处理HemEC,再用ADRB2-hi恢复甚至增加p2-AR的表达,观察裸鼠模型上瘤体一般情况及组织学变化。
结果
1.β2-AR在增殖期血管瘤组织中表达阳性,在静脉畸形和淋巴管畸形组织中表达阴性,β1、β3-AR在增殖期血管瘤、静脉畸形和淋巴管畸形组织中均表达阴性。
2.β2-AR在增殖期血管瘤组织中表达强于消退期血管瘤组织,β1、β3-AR在增殖期和消退期血管瘤组织中无差异。
3.β2-AR在增殖期血管瘤中表达强于其周围正常组织,β1、β3-AR在增殖期血管瘤和其周围正常组织中无差异。
4.β2-AR在HemEC中表达强于HUVEC, β1、β3-AR在HemEC和HUVEC中表达无差异。
5.10μM浓度普萘洛尔治疗HemEC时,细胞增殖能力和成管能力能够得到最大程度的抑制。
6.普萘洛尔和β2-AR特异性抑制剂(ICI)治疗HemEC时,细胞增殖能力和成管能力能够得到最大程度的抑制；β1-AR特异性抑制剂(CGP)能够部分抑制细胞增殖能力和成管能力；β3-AR特异性抑制剂(SR)无抑制细胞增殖和成管的能力。
7.成功设计并构建干扰β2-AR的慢病毒载体,ADRB2-si沉默β2-AR表达,ADRB2-hi增加β2-AR表达,Len-em为空白病毒载体对照。
8.普萘洛尔和ADRB2-si能够通过降低β2-AR的表达来抑制HemEC的增殖能力和成管能力,ADRB2-hi能够通过增加β2-AR的表达来阻断他们的抑制能力。
9.普萘洛尔和ADRB2-si能够通过降低β2-AR的表达来抑制血管瘤模型瘤体生长和瘤体内HemEC的增殖,ADRB2-hi能够通过增加β2-AR的表达来阻断他们的抑制能力。
结论β2-AR在血管瘤发病机制中起到了重要的作用,主要表现在以下几个方面：
1.β2-AR在增殖期血管瘤组织中表达强阳性,在LM和VM等其他血管性良性病变中无明显表达,β1和β3-AR在以上血管性疾病中均无明显表达；由于血管瘤位真性肿瘤,区别于LM和VM等脉管畸形,因此血管瘤的发病机制可能与β2-AR的表达有一定的关系。
2.β2-AR在增殖期血管瘤组织中表达明显高于消退期血管瘤以及瘤体周围正常组织,而β1和β3-AR表达无明显差异；因此p2-AR表达的降低很可能是血管瘤由增殖期转入消退期的原因。
3.β2-AR在增殖期HemEC中表达明显高于对照HUVEC,而β1和p3-AR表达无明显差异；血管瘤来源的HemEC能够形成血管瘤,而正常HUVEC无法成瘤,其重要原因很可能为β2-AR表达的不同。
4.p2-AR被阻断后,HemEC的增殖和成管能力均受到抑制,而β1和p3-AR被阻断后,HemEC的增殖和成管能力无明显变化；因此β2-AR的表达于HemEC的增殖和成管能力有着密切的联系。
5.在体内和体外血管瘤的生长能力都会因为p2-AR的阻断而受到抑制,在重新恢复β2-AR表达后,其生长能力又得到了恢复；因此β2-AR的表达在血管瘤生长过程中起到重要的作用。
Background
Infantile hemangiomas (IH) is a common benign tumor in infants, occurring with high rate8.7%-12.7%from1to12months after born[1-3]. Most IH will regress spontaneously without any scar after earlier proliferative phase. However, some serious IH may affect appearance or even threaten life. Current treatment options include surgery, drug, laser and combination of them. The clinical efficacy is not perfect, because of unclear understanding on pathphysiology and pathogenesis.
There are five clinical characteristics of IH:①Specific natural process:rapid growth of tumor from born and spontaneous regression after one year old.②More female patients:the rate between male and female is1:3-5[5-6]。③Increasing rate on head and neck:the rate of IH on head and neck is60%[7].④More white patients:the white people has greater chance than black and Asian people[8].⑤This disease responds to corticosteroid treatment[9-10].
To investigate the pathphysiology and pathogenesis of IH, it is necessary to establish an animal model. There are five animal models of IH reported as follows:the chicken comb and wattle model[11]; malignant endotheliomas and angiosarcomas nude mice model induced by injections of1,2-DMH[12]; transgenic mice carrying MT gene linked to Py early region regulatory sequences[13]; human hemangioma nude mice model by hemangioma endothelial cell (HemEC) suspension inoculation[14]; human hemangioma nude mice model by tissue block transplantion[15].
Targeting a stable and reliable IH animal model, we compared the feasibility of establishment of human hemangioma animal model by HemEC suspension inoculation and tissue block transplantion.
Objective
1. To isolate and culture hemangioma endothelial cells (HemEC) from fresh infantile hemanioma tissue.
2. To compare the feasibility of establishing human hemangioma animal models by HemEC suspension injection and tissue transplantation.
Methods
1. Specimens of proliferative infantile hemangioma (<3-month-old; untreated) were obtained from Department of Stomotology in Qilu hospital Shandong University and Department of Hemangioma in Linyi tumor hospital. The clinical diagnosis were confirmed in Department of Pathology in Qilu hospital. This study has already got approval from their parents.
2. The tissue was washed by PBS and cut to pieces. Half of the tissue was used for culturing HemEC, the other half was for establishing IH tissue trasplantation mice model. And a few pieces were kept to test the expression of GLUT1by immunohistochemistry (IHC).
3. HemEC was isolated from adherent proliferative IH tissue and tested the expression of vWF and CD34by immunocytochemistry (ICC).
4. BALB/c nude mice (n=12/group) were28±3.2-day-old and20.5±2.1-g-weight. The IH tissue transplantation group:fresh proliferative IH tissue was transplanted into both sides of back or oxter of nude mice; HemEC was injected into both sides of back or oxter of nude mice. The experimental mice were monitored for tumor change and sacrificed to harvest tumor at7,30and60days. The harvested tumor were fixed for HE staining.
Results
1. GLUT1is strongly expressed in obtained tissue, which proves that the tissue is from proliferative IH.
2. The morphology of cultured HemEC is polygon or fusiform. The specific endothelial cells marker vWF and CD34are both positive expressed in HemEC.
3. In the establishment of HemEC injection mice model, all of both sides of12nude mice were successfully tumor-formed. At day7, the tumor was about1mm×1mm×1mm with few new formative vessels; at day30, the tumor was5mm×4mm×3mm with a large number of vasculars surrounded by HemEC; at day60, the tumor was2mm×3mm×2mm with some HemEC apoptosis.
4. In the establishment of IH tissue transplantation mice model, one nude mice was dead by infection, two mice lost their transplanted tissue by lost lines, the other9nude mice were successfully tumor-formed. At day7, the tumor was merged together with body and showed a lot of vasculars; at day30, the tumor was2mm x2mm x2mm with large area of cells apoptosis; at day60, the tumor has already been absorbed by body.
Conclusion
1. It is available to culture HemEC from IH tissue.
2. It is not appropriate to establish IH animal model by IH tissue transplantation.
3. Injection of HemEC in nude mice is a stable and specific IH animal model.
Infantile hemangiomas are the most common benign tumors in infancy. Although natural history and progression of these diseases are well described, their origin remains unclear. Remarkable progress has been gained in the past two decades towards understanding the pathology of these lesions. New studies have created sophisticated hypotheses on the origin of this disease. These include theories of placental origin, somatic endothelial mutation, and extrinsic factors creating an environment for growth. While none of current hypothesis explains all the characteristics of IH, continued research targeting pathology will be helpful for new treatment.
IH is composed of various cell types including hemangioma endothelial cells (HemEC). Recent studies have found that HemEC express P2adrenergic receptors (β2-AR) and β3-AR [1-2]. Propranolol (PRO) is thought to exert its effects on IH progress by three different molecular mechanisms: vasoconstriction, inhibition of angiogenesis and induction of apoptosis.①Both of the autonomic nervous system and epinephrine play a key role in the control of vascular tone. Epinephrine causes either vasoconstriction or vasodilatation by activating α1or β2-AR, respectively.β-AR inhibitors without α-AR antagonistic effects like PRO inhibit epinephrine regulated vasodilatation and this leads to vasoconstriction of HemEC.②During the earlier growth phase of IH, there is increased expression of two major proangogenic factors:basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). Recent publications have reported that the expression of VEGF is reduced by β-AR inhibitors which leads to inhibition of angiogenesis[3].③poptosis is mediated by a lot of pathways, with the β2-AR being the probable receptor involved. It has been hypothesized that by blocking the β2-AR, PRO induces apoptosis at an increased rate[4].
Purpose
1. To detect the expression of β1, β2, β3-AR on infantile hemangioma tissue and HemEC.
2. To observe the function change of HemEC after various specific β1,β2, β3-AR inhibitors treatment.
3. To study the relationship between β2-AR and pathogenesis of IH by lentivirus transfection skill.
Method
1. Fresh tissue of proliferative IH, VM and LM were obtained by surgery. Fix the tissue and test the expression of β1, β32, β3-AR on these tissue by IHC.
2. Isolate RNA and protein from proliferative and involution IH, then detect the expression of β1, β2, β3-AR on RNA and protein level.
3. Isolate RNA and protein from proliferative IH and adjacent tissue, then detect the expression of β1, β2, β3-AR on RNA and protein level.
4. Isolate RNA and protein from HemEC and HUVEC, then detect the expression of β1,β2,(33-AR on RNA and protein level by qPCR and western blotting.
5. HemEC was treated with gradient doses of PRO. The ability of proliferation and tube-formation was detected by MTT and tube-formation assay respectively.
6. HemEC was treated with optimized dose of specific β1,β2,(33-AR inhibitors. The ability of proliferation and tube-formation was detected by MTT and tube-formation assay respectively.
7. Lentivirus combined with β2-AR gene interfere were designed and synthesized. The transfection rate on HemEC was detected by qPCR and western blotting.
8. In vitro, HemEC was treated with PRO or ADRB2-si to knock down expression of β2-AR, and then returned expression of β2-AR by ADRB2-hi transfection. The ability of proliferation and tube-formation was detected by MTT and tube-formation assay respectively.
9. In vivo, the tumor in HemEC injection mice model was treated with PRO or ADRB2-si to knock down expression of P2-AR, and then returned expression of β2-AR by ADRB2-hi transfection. The change of tumor was observed.
Result
1. The expression of β2-AR is positive in proliferative IH, but negative in VM and LM. The expression of (31and β3-AR are negative in proliferative IH,
VM and LM.
2. The expression of β2-AR is higher in proliferative IH than that in involution IH. There are not differences between expression of β1and β3-AR in proliferative and involution IH.
3. The expression of β2-AR is higher in proliferative IH than that in adjacent tissue. There are not differences between expression of β1and β3-AR in proliferative IH and adjacent tissue.
4. The expression of β2-AR is higher in HemEC than that in HUVEC. There are not differences between expression of β1and β3-AR in HemEC and HUVEC.
5. The optimized dose of PRO on affecting proliferation and tube-formation abilities of HemEC is10μM.
6. PRO or ICI (β2-AR specific inhibitor) inhibits the proliferation and tube-formation abilities of HemEC completely. There is partly inhibition from CGP (β1-AR specific inhibitor) and no effect from SR (β3-AR specific inhibitor).
7. By qPCR and WB, ADRB2-si knocks down the expression of β2-AR on HemEC; ADRB2-hi increases the expression of β2-AR on HemEC.
8. PRO and ADRB2-si inhibit the proliferation and tube-formation abilities of HemEC and slow down the tumor growth on IH tumor model, which could be returned by ADRB2-hi.
Conclusion
1. The expression of β2-AR is positive in proliferative IH, but negative in VM and LM. The expression of β1and β3-AR are negative in proliferative IH, VM and LM.
2. The expression of β2-AR is higher in proliferative IH than that in involution IH. There are not differences between expression of β1and β3-AR in proliferative and involution IH.
3. The expression of β2-AR is higher in HemEC than that in HUVEC. There are not differences between expression of β1and β3-AR in HemEC and HUVEC.
4. Blocking β2-AR inhibits the proliferation and tube-formation abilities of HemEC completely. There is partly inhibition from blocking (31-AR and no effect from blocking β3-AR.
5. Blocking β2-AR reduces the tumor-forming on HemEC injection mice model, and recover the expression of (32-AR can reverse it.
婴幼儿血管瘤(infantile hemangioma, IH)是多发于婴幼儿的一种良性肿瘤,出生后1个月到1岁内发病率高达8.7%-12.7%[1-3],影响患儿的外貌,部分严重者会涉及功能性器官,甚至危及生命。目前对该病的治疗主要采取手术、药物、激光等措施,或采用联合治疗的方案,但山于缺少对血管瘤病因和病理机制的认识,治疗效果不能令人满意[4]。
血管瘤的发病具有以下5个临床特点：①出生后即发病,1个月到1岁内发展迅速；1岁以后病变稳定并开始自发性消退,有着明显的迅速增殖期和自发性消退期的表现。②患者以女性居多,男女比率约为1：3-5[5-6]。③头颈部血管瘤约占全身发病的60%[7]。④白人罹患率是黑种人或黄种人的10-12倍[8]。⑤适当的激素治疗对该病有效[9-10]。
研究血管瘤发病机制的首要问题是建立符合IH病变自然进程的动物模型。目前报道过的IH动物模型建立方法主要有5种：①采用鸡冠和肉垂等富含血窦组织制备的皮肤毛细血管瘤模型[11]；②以化学药物如1,2-二甲肼(1,2-dimethylhydrazine dihydrochloride,1,2-DMH)诱发的恶性内皮细胞瘤或血管肉瘤[12]；③通过转基因方法将多瘤病毒(polyomavirus, Py)的中等T抗原(middle T antigen, MT)基因整合到小鼠正常基因序列的恶性多瘤病毒模型[13]；④将体外培养的血管瘤内皮细胞(hemangioma endothelial cell, HemEC)注射入裸鼠(免疫缺陷小鼠)皮下形成血管瘤[14]；⑤裸鼠皮下植入新鲜增殖期血管瘤组织[15]。
本课题针对以上模型中报道最多的HemEC皮下注射裸鼠模型和增殖期血管瘤组织块皮下移植裸鼠模型进行了比较,以选择最稳定、可靠的血管瘤动物模型,为接下来血管瘤发病机制的研究打下基础。
目的
1.探讨体外培养HemEC的可行性。
2.比较HemEC注射法和血管瘤组织块植入法裸鼠模型与血管瘤自然进程的相似性以及在血管瘤动物模型建立中的稳定性。
方法
1.收集山东大学齐鲁医院口腔颌面外科和临沂市肿瘤医院血管瘤科的血管瘤患者组织标本,取样条件为年龄小于3个月并且未接受任何治疗的2例手术切除病例。本研究已告知患儿监护人并签署知情同意书。
2.冲洗新鲜婴幼儿血管瘤组织块并修剪成1cm3大小,50%用于HemEC培养,50%用于构建组织块植入法裸鼠皮下血管瘤模型；另取少量组织固定,石蜡包埋后,免疫组织化学法(immunohistochemistry, IHC)鉴定葡萄糖转运蛋白1(glucose transporter1,GLUT1)的表达。
3.HemEC体外培养及鉴定：利用组织块贴壁培养法分离并培养HemEC;免疫细胞化学法(immunocytochemistry, ICC)检测内皮细胞标记物第八因子相关抗原因子(factor Ⅷ-related antigen, von Willebrand factor, vWF)和CD34在HemEC上的表达。
4.实验用裸鼠(BALB/c nude mice)24只,雌雄不限,鼠龄(28+3.2)d,体重(20.5+2.1)g,购白北京华阜康生物科技股份公司。24只裸鼠随机分为2组：组织块组和细胞注射组,每组分别12只。组织块组：双侧腋窝皮下植入处理过的1cm3大小组织块；细胞组：双侧腋窝皮下注射第3代HemEC。分别记录第7、30、60d两组血管瘤模型移植处瘤体的变化,包括瘤体尺寸、色泽和质地；在各时间点各组随机选取4只裸鼠切取移植处瘤体组织,固定,石蜡包埋后,苏木素-伊红(hematoxylin-eosin staining, HE)染色,镜下观察其组织学特点。
结果
1.IHC法鉴定GLUT1的表达检测血管瘤特异性标记物GLUT1在手术取得组织中的表达为强阳性,证明为增殖期血管瘤组织：HemEC皮下注射法血管瘤裸鼠模型第30d时,瘤体组织GLUT1呈阳性表达。
2.HemEC的培养及鉴定HemEC在组织块培养法下生长并传代,细胞贴壁生长,形状不规则,多为多角形或梭形,聚集生长；ICC法检测内皮细胞标记物vWF和CD34,均呈阳性表达。
3.HemEC皮下注射法血管瘤裸鼠模型的建立在12只裸鼠双侧腋下部皮下组织注射HemEC后,双侧全部成瘤；第7d时,瘤体约1mm×1mm×1mm大小,镜下见大量HemEC,并逐渐开始形成血管；第30d时,瘤体生长至5mm×4mm×3mm大小,镜下见大量密集生长的HemEC,并有大量HemEC围成的小血管,局部可见大血管；第60d时,瘤体为2mm×3mm×2mm大小,部分HemEC开始逐渐凋亡,血管萎缩或破裂。
4.增殖划血管瘤组织块移植法血管瘤裸鼠模型的建立移植组织块到裸鼠双侧腋下部皮下后,1周之内便有1只裸鼠因感染死亡,2只裸鼠因缝线被撕咬或挠抓而过早脱落,导致组织块从体内排出,其余9只裸鼠均双侧腋下成瘤。第7d时,移植块已与机体融合,镜下见HemEC围绕的密集小血管；第30d时,瘤体约2mm×2mm×2mm,镜下见大量移植细胞的变性、坏死,炎细胞遍布,仅有少量血管残留；第60d时,移植组织已完全融于机体,镜下可见移植物已脂肪化,仅见少量血管周围有类似残留移植物存在。
结论
1.血管瘤组织块贴壁法可以培养出HemEC。
2.组织块移植法血管瘤裸鼠模型因高失败率和无法大量建模的缺点,不能为本课题所用。
3.皮下注射人血管瘤内皮细胞的血管瘤裸鼠模型建模方法因其稳定性高,特异性强和可重复性的优点,是建立血管瘤的动物模型较为较理想的选择。
婴幼儿血管瘤(infantile hemangiomas, IH)是最常见的婴幼儿良性肿瘤之一,尽管对其组织学特点和发病机制的研究众多,但是一直没有将这些问题解释清楚。过去的几十年中,大量科研工作者致力于对该疾病发病机制的研究,也取得了一些进展,并建立了一些相应的学说,如胎盘绒毛膜学说,内皮细胞基因突变,以及外源性因子形成促进细胞生长的微环境等。但是仍然没有一种学说能够完全解释IH所有的临床和生理特征,因此需要一些突破性的发现来帮助我们理解并研究IH的发病机制,从而能够研发出对IH特异性更强的治疗方案。
口服普萘洛尔(一种非选择性beta1肾上腺素受体抑制剂)被报道能够有效治疗血管瘤,这一新发现使众多研究者将目光转移到了beta1肾上腺素受体(βadrenergic receptor, β-AR)与血管瘤之间的关系上。血管瘤由以血管瘤内皮细胞(hemangioma endothelial cell, HemEC)为主的多种细胞组成,p2-AR可在内皮细胞表面表达[1],而在HemEC上也发现了有β2和β3-AR的表达[2]。因此我们假设,普萘洛尔发挥治疗血管瘤的作用机制可能是通过以下3个方面实现的：①早期血管收缩。自主神经系统和肾上腺素在皿管管腔收缩与舒张的控制上起着至关重要的作用,肾上腺素依靠激活a1和β2-AR能够控制血管的收缩和舒张,所以单独的p2-AR抑制剂(如普萘洛尔)则可以阻断肾上腺素激活β2-AR引起的血管舒张,并且避免α1-AR被抑制,保留了对血管收缩的刺激。②中期抑制血管生成。在血管瘤增殖发育的旺盛阶段,有两个促血管生成因子的表达异常增高：血管内皮细胞生长因子(vascular endothelial growth factor, VEGF)和成纤维细胞生长因子(basic fibroblast growth factor, bFGF),在β2-AR抑制剂的阻断作用下,减少的VEGF分泌可有效的抑制血管生成[3]。③长期诱导HemEC的凋亡。HemEC的凋亡被多条信号通路所介导,其中β2-AR被抑制而诱发的下游信号通路所介导的细胞凋亡率最高[4]。
验证以上假说的关键是确定β2-AR在血管瘤发病机制中的重要作用,包括β2-AR的表达以及β2-AR表达被干扰后血管瘤症状的改变。以β2-AR为始点,对其下游信号通路的研究将是我们今后工作的重点。
目的
1.检测p-AR各亚型在血管瘤以及血管瘤内皮细胞中的表达情况。
2.观察β-AR各亚型被分别抑制后血管瘤发病情况的改变。
3.利用病毒转染携带干扰基因,探讨p2-AR在体内和体外血管瘤发病机制中的作用。
方法
1.获取新鲜增殖气期血管瘤、静脉畸形(vein malformation, VM)和淋巴管畸形(lymphatic malformation, LM)组织,固定包埋,制作切片,免疫组化法(immunohistochemistry, IHC)检测β1、β2、β3-AR的表达。
2.获取新鲜增殖期和消退期血管瘤组织,提取RNA和蛋白,qPCR和WB法检测β1、β2、p3-AR的表达。
3.获取新鲜增殖期血管瘤及其周围正常组织,提取RNA和蛋白,qPCR和WB法检测β1、β2、β3-AR的表达。
4.组织块法培养HemEC,以脐静脉内皮细胞(HUVEC)为对照,提取RNA和蛋白,qPCR和WB法检测β1、 β2、β3-AR的表达。
5.在体外,以梯度浓度普萘洛尔治疗HemEC, MTT法检测细胞增殖能力,成管实验检测细胞成管能力,经对比得到普萘洛尔治疗HemEC的最佳浓度。
6.在体外,用最佳浓度的p-AR各亚型特异性抑制剂治疗HemEC, MTT法检测细胞增殖能力,成管实验检测细胞成管能力,得到与普萘洛尔作用最接近的抑制剂所属的p-AR亚型。
7.设计并构建特异性干扰p2-AR慢病毒载体,转染HemEC,并提取RNA和蛋白,qPCR和WB法检测β2-AR的表达,以确定其病毒转染率。
8.在体外,用普萘洛尔和沉默β2-AR慢病毒(ADRB2-si)处理HemEC,再用高表达β2-AR慢病毒(ADRB2-hi)恢复甚至增加β2-AR的表达,MTT法检测细胞增殖能力,成管实验检测细胞成管能力的变化。
9.在HemEC皮下注射血管瘤模型上,用普萘洛尔和ADRB2-si处理HemEC,再用ADRB2-hi恢复甚至增加p2-AR的表达,观察裸鼠模型上瘤体一般情况及组织学变化。
结果
1.β2-AR在增殖期血管瘤组织中表达阳性,在静脉畸形和淋巴管畸形组织中表达阴性,β1、β3-AR在增殖期血管瘤、静脉畸形和淋巴管畸形组织中均表达阴性。
2.β2-AR在增殖期血管瘤组织中表达强于消退期血管瘤组织,β1、β3-AR在增殖期和消退期血管瘤组织中无差异。
3.β2-AR在增殖期血管瘤中表达强于其周围正常组织,β1、β3-AR在增殖期血管瘤和其周围正常组织中无差异。
4.β2-AR在HemEC中表达强于HUVEC, β1、β3-AR在HemEC和HUVEC中表达无差异。
5.10μM浓度普萘洛尔治疗HemEC时,细胞增殖能力和成管能力能够得到最大程度的抑制。
6.普萘洛尔和β2-AR特异性抑制剂(ICI)治疗HemEC时,细胞增殖能力和成管能力能够得到最大程度的抑制；β1-AR特异性抑制剂(CGP)能够部分抑制细胞增殖能力和成管能力；β3-AR特异性抑制剂(SR)无抑制细胞增殖和成管的能力。
7.成功设计并构建干扰β2-AR的慢病毒载体,ADRB2-si沉默β2-AR表达,ADRB2-hi增加β2-AR表达,Len-em为空白病毒载体对照。
8.普萘洛尔和ADRB2-si能够通过降低β2-AR的表达来抑制HemEC的增殖能力和成管能力,ADRB2-hi能够通过增加β2-AR的表达来阻断他们的抑制能力。
9.普萘洛尔和ADRB2-si能够通过降低β2-AR的表达来抑制血管瘤模型瘤体生长和瘤体内HemEC的增殖,ADRB2-hi能够通过增加β2-AR的表达来阻断他们的抑制能力。
结论β2-AR在血管瘤发病机制中起到了重要的作用,主要表现在以下几个方面：
1.β2-AR在增殖期血管瘤组织中表达强阳性,在LM和VM等其他血管性良性病变中无明显表达,β1和β3-AR在以上血管性疾病中均无明显表达；由于血管瘤位真性肿瘤,区别于LM和VM等脉管畸形,因此血管瘤的发病机制可能与β2-AR的表达有一定的关系。
2.β2-AR在增殖期血管瘤组织中表达明显高于消退期血管瘤以及瘤体周围正常组织,而β1和β3-AR表达无明显差异；因此p2-AR表达的降低很可能是血管瘤由增殖期转入消退期的原因。
3.β2-AR在增殖期HemEC中表达明显高于对照HUVEC,而β1和p3-AR表达无明显差异；血管瘤来源的HemEC能够形成血管瘤,而正常HUVEC无法成瘤,其重要原因很可能为β2-AR表达的不同。
4.p2-AR被阻断后,HemEC的增殖和成管能力均受到抑制,而β1和p3-AR被阻断后,HemEC的增殖和成管能力无明显变化；因此β2-AR的表达于HemEC的增殖和成管能力有着密切的联系。
5.在体内和体外血管瘤的生长能力都会因为p2-AR的阻断而受到抑制,在重新恢复β2-AR表达后,其生长能力又得到了恢复；因此β2-AR的表达在血管瘤生长过程中起到重要的作用。
Background
Infantile hemangiomas (IH) is a common benign tumor in infants, occurring with high rate8.7%-12.7%from1to12months after born[1-3]. Most IH will regress spontaneously without any scar after earlier proliferative phase. However, some serious IH may affect appearance or even threaten life. Current treatment options include surgery, drug, laser and combination of them. The clinical efficacy is not perfect, because of unclear understanding on pathphysiology and pathogenesis.
There are five clinical characteristics of IH:①Specific natural process:rapid growth of tumor from born and spontaneous regression after one year old.②More female patients:the rate between male and female is1:3-5[5-6]。③Increasing rate on head and neck:the rate of IH on head and neck is60%[7].④More white patients:the white people has greater chance than black and Asian people[8].⑤This disease responds to corticosteroid treatment[9-10].
To investigate the pathphysiology and pathogenesis of IH, it is necessary to establish an animal model. There are five animal models of IH reported as follows:the chicken comb and wattle model[11]; malignant endotheliomas and angiosarcomas nude mice model induced by injections of1,2-DMH[12]; transgenic mice carrying MT gene linked to Py early region regulatory sequences[13]; human hemangioma nude mice model by hemangioma endothelial cell (HemEC) suspension inoculation[14]; human hemangioma nude mice model by tissue block transplantion[15].
Targeting a stable and reliable IH animal model, we compared the feasibility of establishment of human hemangioma animal model by HemEC suspension inoculation and tissue block transplantion.
Objective
1. To isolate and culture hemangioma endothelial cells (HemEC) from fresh infantile hemanioma tissue.
2. To compare the feasibility of establishing human hemangioma animal models by HemEC suspension injection and tissue transplantation.
Methods
1. Specimens of proliferative infantile hemangioma (<3-month-old; untreated) were obtained from Department of Stomotology in Qilu hospital Shandong University and Department of Hemangioma in Linyi tumor hospital. The clinical diagnosis were confirmed in Department of Pathology in Qilu hospital. This study has already got approval from their parents.
2. The tissue was washed by PBS and cut to pieces. Half of the tissue was used for culturing HemEC, the other half was for establishing IH tissue trasplantation mice model. And a few pieces were kept to test the expression of GLUT1by immunohistochemistry (IHC).
3. HemEC was isolated from adherent proliferative IH tissue and tested the expression of vWF and CD34by immunocytochemistry (ICC).
4. BALB/c nude mice (n=12/group) were28±3.2-day-old and20.5±2.1-g-weight. The IH tissue transplantation group:fresh proliferative IH tissue was transplanted into both sides of back or oxter of nude mice; HemEC was injected into both sides of back or oxter of nude mice. The experimental mice were monitored for tumor change and sacrificed to harvest tumor at7,30and60days. The harvested tumor were fixed for HE staining.
Results
1. GLUT1is strongly expressed in obtained tissue, which proves that the tissue is from proliferative IH.
2. The morphology of cultured HemEC is polygon or fusiform. The specific endothelial cells marker vWF and CD34are both positive expressed in HemEC.
3. In the establishment of HemEC injection mice model, all of both sides of12nude mice were successfully tumor-formed. At day7, the tumor was about1mm×1mm×1mm with few new formative vessels; at day30, the tumor was5mm×4mm×3mm with a large number of vasculars surrounded by HemEC; at day60, the tumor was2mm×3mm×2mm with some HemEC apoptosis.
4. In the establishment of IH tissue transplantation mice model, one nude mice was dead by infection, two mice lost their transplanted tissue by lost lines, the other9nude mice were successfully tumor-formed. At day7, the tumor was merged together with body and showed a lot of vasculars; at day30, the tumor was2mm x2mm x2mm with large area of cells apoptosis; at day60, the tumor has already been absorbed by body.
Conclusion
1. It is available to culture HemEC from IH tissue.
2. It is not appropriate to establish IH animal model by IH tissue transplantation.
3. Injection of HemEC in nude mice is a stable and specific IH animal model.
Infantile hemangiomas are the most common benign tumors in infancy. Although natural history and progression of these diseases are well described, their origin remains unclear. Remarkable progress has been gained in the past two decades towards understanding the pathology of these lesions. New studies have created sophisticated hypotheses on the origin of this disease. These include theories of placental origin, somatic endothelial mutation, and extrinsic factors creating an environment for growth. While none of current hypothesis explains all the characteristics of IH, continued research targeting pathology will be helpful for new treatment.
IH is composed of various cell types including hemangioma endothelial cells (HemEC). Recent studies have found that HemEC express P2adrenergic receptors (β2-AR) and β3-AR [1-2]. Propranolol (PRO) is thought to exert its effects on IH progress by three different molecular mechanisms: vasoconstriction, inhibition of angiogenesis and induction of apoptosis.①Both of the autonomic nervous system and epinephrine play a key role in the control of vascular tone. Epinephrine causes either vasoconstriction or vasodilatation by activating α1or β2-AR, respectively.β-AR inhibitors without α-AR antagonistic effects like PRO inhibit epinephrine regulated vasodilatation and this leads to vasoconstriction of HemEC.②During the earlier growth phase of IH, there is increased expression of two major proangogenic factors:basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). Recent publications have reported that the expression of VEGF is reduced by β-AR inhibitors which leads to inhibition of angiogenesis[3].③poptosis is mediated by a lot of pathways, with the β2-AR being the probable receptor involved. It has been hypothesized that by blocking the β2-AR, PRO induces apoptosis at an increased rate[4].
Purpose
1. To detect the expression of β1, β2, β3-AR on infantile hemangioma tissue and HemEC.
2. To observe the function change of HemEC after various specific β1,β2, β3-AR inhibitors treatment.
3. To study the relationship between β2-AR and pathogenesis of IH by lentivirus transfection skill.
Method
1. Fresh tissue of proliferative IH, VM and LM were obtained by surgery. Fix the tissue and test the expression of β1, β32, β3-AR on these tissue by IHC.
2. Isolate RNA and protein from proliferative and involution IH, then detect the expression of β1, β2, β3-AR on RNA and protein level.
3. Isolate RNA and protein from proliferative IH and adjacent tissue, then detect the expression of β1, β2, β3-AR on RNA and protein level.
4. Isolate RNA and protein from HemEC and HUVEC, then detect the expression of β1,β2,(33-AR on RNA and protein level by qPCR and western blotting.
5. HemEC was treated with gradient doses of PRO. The ability of proliferation and tube-formation was detected by MTT and tube-formation assay respectively.
6. HemEC was treated with optimized dose of specific β1,β2,(33-AR inhibitors. The ability of proliferation and tube-formation was detected by MTT and tube-formation assay respectively.
7. Lentivirus combined with β2-AR gene interfere were designed and synthesized. The transfection rate on HemEC was detected by qPCR and western blotting.
8. In vitro, HemEC was treated with PRO or ADRB2-si to knock down expression of β2-AR, and then returned expression of β2-AR by ADRB2-hi transfection. The ability of proliferation and tube-formation was detected by MTT and tube-formation assay respectively.
9. In vivo, the tumor in HemEC injection mice model was treated with PRO or ADRB2-si to knock down expression of P2-AR, and then returned expression of β2-AR by ADRB2-hi transfection. The change of tumor was observed.
Result
1. The expression of β2-AR is positive in proliferative IH, but negative in VM and LM. The expression of (31and β3-AR are negative in proliferative IH,
VM and LM.
2. The expression of β2-AR is higher in proliferative IH than that in involution IH. There are not differences between expression of β1and β3-AR in proliferative and involution IH.
3. The expression of β2-AR is higher in proliferative IH than that in adjacent tissue. There are not differences between expression of β1and β3-AR in proliferative IH and adjacent tissue.
4. The expression of β2-AR is higher in HemEC than that in HUVEC. There are not differences between expression of β1and β3-AR in HemEC and HUVEC.
5. The optimized dose of PRO on affecting proliferation and tube-formation abilities of HemEC is10μM.
6. PRO or ICI (β2-AR specific inhibitor) inhibits the proliferation and tube-formation abilities of HemEC completely. There is partly inhibition from CGP (β1-AR specific inhibitor) and no effect from SR (β3-AR specific inhibitor).
7. By qPCR and WB, ADRB2-si knocks down the expression of β2-AR on HemEC; ADRB2-hi increases the expression of β2-AR on HemEC.
8. PRO and ADRB2-si inhibit the proliferation and tube-formation abilities of HemEC and slow down the tumor growth on IH tumor model, which could be returned by ADRB2-hi.
Conclusion
1. The expression of β2-AR is positive in proliferative IH, but negative in VM and LM. The expression of β1and β3-AR are negative in proliferative IH, VM and LM.
2. The expression of β2-AR is higher in proliferative IH than that in involution IH. There are not differences between expression of β1and β3-AR in proliferative and involution IH.
3. The expression of β2-AR is higher in HemEC than that in HUVEC. There are not differences between expression of β1and β3-AR in HemEC and HUVEC.
4. Blocking β2-AR inhibits the proliferation and tube-formation abilities of HemEC completely. There is partly inhibition from blocking (31-AR and no effect from blocking β3-AR.
5. Blocking β2-AR reduces the tumor-forming on HemEC injection mice model, and recover the expression of (32-AR can reverse it.
引文
1 Amir J, Metzker A, Krikler R, et al. Strawberry hemangioma in preterm infants. Pediatr Dermatol 1986; 3(4):331-2.
2 Bivings L. Spontaneous regression of angiomas in children; twenty-two years' observation covering 236 cases. J Pediatr 1954; 45(6):643-7.
3 Jacobs AH. Strawberry hemangiomas; the natural history of the untreated lesion. Calif Med 1957; 86(1):8-10.
4 郑家伟,杨秀娟.血管瘤的治疗选择.中国实用口腔科杂志 2009：2(5)：274-9.
5 Enjolras O and Mulliken JB. The current management of vascular birthmarks. Pediatr Dermatol 1993; 10(4):311-3.
6 Margileth AM and Museles M. Cutaneous hemangiomas in children. Diagnosis and conservative management. Jama 1965; 194(5):523-6.
7 Robertson RL, Robson CD, Barnes PD, et al. Head and neck vascular anomalies of childhood. Neuroimaging Clin NAm 1999; 9(1):115-32.
8 Rutledge DR. Are there beta-adrenergic receptor response differences between racial groups? Dicp 1991; 25(7-8):824-34.
9 Holland KE and Drolet BA. Infantile hemangioma. Pediatr Clin North Am 2010; 57(5): 1069-83.
10 Jia J and Zhao YF. Biomarkers:important clues to the pathogenesis of infantile haemangioma and their clinical significance. Chin J Dent Res 2010; 13(2):105-8.
11 Ritter EJ, Goldman L, Richfield D, et al. The chicken comb and wattle as experimental model for investigative argon laser therapy of angiomas. Acta Derm Venereol 1969; 49(3): 304-8.
12 Sato N, Sato T, Takahashi S, et al. Establishment of murine endothelial cell lines that develop angiosarcomas in vivo:brief demonstration of a proposed animal model for Kaposi's sarcoma. Cancer Res 1986; 46(1):362-6.
13 Bautch VL, Toda S, Hassell JA, et al. Endothelial cell tumors develop in transgenic mice carrying polyoma virus middle Toncogene. Cell 1987; 51(4):529-37.
14 虎小毅,杨壮群,宋勇吗,等.细胞悬液接种法血管瘤裸鼠动物模型的建立.中国美容医学2008；17(7)：1012-4.
15 Tang Y, Liu W, Yu S, et al. A novel in vivo model of human hemangioma: xenograft of human hemangioma tissue on nude mice. Plast Reconstr Surg 2007; 120(4):869-78.
16 Hohenleutner U, Landthaler M, Hamm H, et al. Hemangiomas of infancy and childhood. J Dtsch Dermatol Ges 2007; 5(4):334-8.
17 Akhavan A and Zippin JH. Current treatments for infantile hemangiomas. J Drugs Dermatol 2010; 9(2):176-80.
18 Berard M, Sordello S, Ortega N, et al. Vascular endothelial growth factor confers a growth advantage in vitro and in vivo to stromal cells cultured from neonatal hemangiomas. Am J Pathol 1997; 150(4):1315-26.
19 Chang J, Most D, Bresnick S, et al. Proliferative hemangiomas:analysis of cytokine gene expression and angiogenesis. Plast Reconstr Surg 1999; 103(1):1-9; discussion 10.
20 Giatromanolaki A, Arvanitidou V, Hatzimichael A, et al. The HIF-2alpha/VEGF pathway activation in cutaneous capillary haemangiomas. Pathology 2005; 37(2):149-51.
21 Kleinman ME, Greives MR, Churgin SS, et al. Hypoxia-induced mediators of stem/progenitor cell trafficking are increased in children with hemangioma. Arterioscler Thromb Vase Biol 2007; 27(12):2664-70.
22 Qu Z, Liebler JM, Powers MR, et al. Mast cells are a major source of basic fibroblast growth factor in chronic inflammation and cutaneous hemangioma. Am J Pathol 1995; 147(3):564-73.
23 Takahashi K, Mulliken JB, Kozakewich HP, et al. Cellular markers that distinguish the phases of hemangioma during infancy and childhood. J Clin Invest 1994; 93(6):2357-64.
24 Mulliken JB, Fishman SJ and Burrows PE. Vascular anomalies. Curr Probl Surg 2000; 37(8):517-84.
25 Schwartz RA, Sidor Ml, Musumeci ML, et al. Infantile haemangiomas:a challenge in paediatric dermatology. J Eur Acad Dermatol Venereol 2010; 24(6):631-8.
26 Soldatskii lu L, Shekhter AB, Ponkratenko AD, et al. [Effect of cryotherapy and laser destruction on experimental model of human vascular tumor]. Vestn Otorinolaringol 1995(2):10-4.
27 Anvari B, Tanenbaum BS, Hoffman W, et al. Nd:YAG laser irradiation in conjunction with cryogen spray cooling induces deep and spatially selective photocoagulation in animal models. Phys Med Biol 1997; 42(2):265-82.
28 Softer D, Resnick-Roguel N, Eldor A, et al. Multifocal vascular tumors in fowl induced by a newly isolated retrovirus. Cancer Res 1990; 50(15):4787-93.
29 Liekens S, Andrei G, Vandeputte M, et al. Potent inhibition of hemangioma formation in rats by the acyclic nucleoside phosphonate analogue cidofovir. Cancer Res 1998; 58(12): 2562-7.
30 Taraboletti G, Belotti D, Dejana E, et al. Endothelial cell migration and invasiveness are induced by a soluble factor produced by murine endothelioma cells transformed by polyoma virus middle T oncogene. Cancer Res 1993; 53(16):3812-6.
31 Kiefer F, Anhauser I, Soriano P, et al. Endothelial cell transformation by polyomavirus middle T antigen in mice lacking Src-related kinases. Curr Biol 1994; 4(2):100-9.
32 Lee RJ, Springer ML, Blanco-Bose WE, et al. VEGF gene delivery to myocardium: deleterious effects of unregulated expression. Circulation 2000; 102(8):898-901.
33 Flanagan SP.'Nude', a new hairless gene with pleiotropic effects in the mouse. Genet Res 1966; 8(3):295-309.
34 Povlsen CO. Heterotransplants of human tumors in nude mice. Antibiot Chemother 1980; 28:15-20.
35 Sharkey FE and Fogh J. Considerations in the use of nude mice for cancer research. Cancer Metastasis Rev 1984; 3(4):341-60.
36 van Weerden WM and Romijn JC. Use of nude mouse xenograft models in prostate cancer research. Prostate 2000; 43(4):263-71.
37 Valerio MG, Fineman EL, Bowman RL, et al. Long-term survival of normal adult human tissues as xenografts in congenially athymic nude mice. J Natl Cancer Inst 1981; 66(5): 849-58.
38 Kischer CW, Sheridan D and Pindur J. Use of nude (athymic) mice for the study of hypertrophic scars and keloids:vascular continuity between mouse and implants. Anat Rec 1989; 225(3):189-96.
39 Gilhar A and Etzioni A. The nude mouse model for the study of human skin disorders. Dermatology 1994; 189(1):5-8.
40 Gerber H, Wagner HE, Burgi U, et al. Model of the athymic nude mouse for the study of benign goiter disease. Exp Clin Endocrinol Diabetes 1996; 104 Suppl 3:56-9.
41 Pouliot R, Larouche D, Auger FA, et al. Reconstructed human skin produced in vitro and grafted on athymic mice. Transplantation 2002; 73(11):1751-7.
42 Honaramooz A, Snedaker A, Boiani M, et al. Sperm from neonatal mammalian testes grafted in mice. Nature 2002; 418(6899):778-81.
43 Guerret S, Govignon E, Hartmann DJ, et al. Long-term remodeling of a bilayered living human skin equivalent (Apligraf) grafted onto nude mice:immunolocalization of human cells and characterization of extracellular matrix. Wound Repair Regen 2003; 11(1): 35-45.
44 Martin-Padura I, De Castellarnau C, Uccini S, et al. Expression of VE (vascular endothelial)-cadherin and other endothelial-specific markers in haemangiomas. J Pathol 1995; 175(1):51-7.
45 Dosanjh A, Chang J, Bresnick S, et al. In vitro characteristics of neonatal hemangioma endothelial cells:similarities and differences between normal neonatal and fetal endothelial cells. J Cutan Pathol 2000; 27(9):441-50.
46赵曜,谭玉珍,周良辅,等.中枢神经系统海绵状血管瘤内皮细胞的分离和培养.中华实验外科杂志2003;20(8)：685-6
47 Gould GW and Holman GD. The glucose transporter family:structure, function and tissue-specific expression. Biochem J 1993; 295 (Pt2):329-41.
48 Jansson T, Wennergren M and Powell TL Placental glucose transport and GLUT 1 expression in insulin-dependent diabetes. Am J Obstet Gynecol 1999; 180(1 Pt 1):163-8.
49 Schlingemann RO, Hofman P, Klooster J, et al. Ciliary muscle capillaries have blood-tissue barrier characteristics. Exp Eye Res 1998; 66(6):747-54.
50 North PE, Waner M, Mizeracki A, et al. GLUT1:a newly discovered immunohistochemical marker for juvenile hemangiomas. Hum Pathol 2000; 31(1):11-22.
51 Mo JQ, Dimashkieh HH and Bove KE. GLUT1 endothelial reactivity distinguishes hepatic infantile hemangioma from congenital hepatic vascular malformation with associated capillary proliferation. Hum Pathol 2004; 35(2):200-9.
52 Lo K, Mihm M and Fay A. Current theories on the pathogenesis of infantile hemangioma. Semin Ophthalmol 2009; 24(3):172-7.
53 Barnes CM, Huang S, Kaipainen A, et al. Evidence by molecular profiling for a placental origin of infantile hemangioma. Proc Natl Acad Sci U S A 2005; 102(52):19097-102.
54 North PE, Waner M, Mizeracki A, et al. A unique microvascular phenotype shared by juvenile hemangiomas and human placenta. Arch Dermatoi 2001; 137(5):559-70.
55 Khan ZA, Boscolo E, Picard A, et al. Multipotential stem cells recapitulate human infantile hemangioma in immunodeficient mice. J Clin Invest 2008; 118(7):2592-9.
1 laccarino G, Ciccarelli M, Sorriento D, et al. Ischemic neoangiogenesis enhanced by beta2-adrenergic receptor overexpression:a novel role for the endothelial adrenergic system. Circ Res 2005; 97(11):1182-9.
2 Chisholm KM, Chang KW, Truong MT, et al. beta-Adrenergic receptor expression in vascular tumors. Mod Pathol 2012.
3 Fredriksson JM, Lindquist JM, Bronnikov GE, et al. Norepinephrine induces vascular endothelial growth factor gene expression in brown adipocytes through a beta-adrenoreceptor/cAMP/protein kinase A pathway involving Src but independently of Erk1/2. J Biol Chem 2000; 275(18):13802-11.
4 Zhang D, Ma Q, Shen S, et al. Inhibition of pancreatic cancer cell proliferation by propranolol occurs through apoptosis induction:the study of beta-adrenoceptor antagonist's anticancer effect in pancreatic cancer cell. Pancreas 2009; 38(1):94-100.
5 Jia J and Zhao YF. Biomarkers:important clues to the pathogenesis of infantile haemangioma and their clinical significance. Chin J Dent Res 2010; 13(2):105-8.
6 Holland KE and Drolet BA. Infantile hemangioma. Pediatr Clin North Am 2010; 57(5): 1069-83.
7 郑家伟,杨秀娟.血管瘤的治疗选择.中国实用口腔科杂志2009;2(5)：274-9.
8 Mulliken JB, Fishman SJ and Burrows PE. Vascular anomalies. Curr Probl Surg 2000; 37(8):517-84.
9 Waner M and Suen JY. Management of congenital vascular lesions of the head and neck. Oncology (Williston Park) 1995; 9(10):989-94,97; discussion 98 passim.
10 Mulliken JB and Glowacki J. Classification of pediatric vascular lesions. Plast Reconstr Surg 1982; 70(1):120-1.
11 Hohenleutner U, Landthaler M, Hamm H, et al. Hemangiomas of infancy and childhood. J Dtsch Dermatol Ges 2007; 5(4):334-8.
12 Storch CH and Hoeger PH. Propranolol for infantile haemangiomas:insights into the molecular mechanisms of action. Br J Dermatol 2010; 163(2):269-74.
13 Akhavan A and Zippin JH. Current treatments for infantile hemangiomas. J Drugs Dermatol 2010; 9(2):176-80.
14仇雅璟,马刚,林晓曦.婴幼儿血管瘤发生与形成机制的研究进展.中华整形外科杂志2012;28(3)：233-6.
15 Boye E, Yu Y, Paranya G, et al. Clonality and altered behavior of endothelial cells from hemangiomas. J Clin Invest 2001; 107(6):745-52.
16 Barnes CM, Huang S, Kaipainen A, et al. Evidence by molecular profiling for a placental origin of infantile hemangioma. Proc Natl Acad Sci U S A 2005; 102(52):19097-102.
17 Khan ZA, Boscolo E, Picard A, et al. Multipotential stem cells recapitulate human infantile hemangioma in immunodeficient mice. J Clin Invest 2008; 118(7):2592-9.
18 North PE, Waner M, Mizeracki A, et al. A unique microvascular phenotype shared by juvenile hemangiomas and human placenta. Arch Dermatol 2001; 137(5):559-70.
19 Leaute-Labreze C, Dumas de la Roque E, Hubiche T, et al. Propranolol for severe hemangiomas of infancy. N Engl J Med 2008; 358(24):2649-51.
20 Buckmiller LM, Munson PD, Dyamenahalli U, et al. Propranolol for infantile hemangiomas: early experience at a tertiary vascular anomalies center. Laryngoscope 2010; 120(4): 676-81.
21 Sans V, Dumas de la Roque E, Berge J, et al. Propranolol for Severe Infantile Hemangiomas:Follow-Up Report. Pediatrics 2009.
22 Denoyelle F, Leboulanger N, Enjolras O, et al. Role of Propranolol in the therapeutic strategy of infantile laryngotracheal hemangioma. Int J Pediatr Otorhinolaryngol 2009; 73(8):1168-72.
23 Rosbe KW, Suh KY, Meyer AK, et al. Propranolol in the management of airway infantile hemangiomas. Arch Otolaryngol Head Neck Surg 2010; 136(7):658-65.
24 Marsciani A, Pericoli R, Alaggio R, et al. Massive response of severe infantile hepatic hemangioma to propanolol. Pediatr Blood Cancer 2010; 54(1):176.
25 Li YC, McCahon E, Rowe NA, et al. Successful treatment of infantile haemangiomas of the orbit with propranolol. Clin Experiment Ophthalmol 2010; 38(6):554-9.
26 Zheng M, Zhu W, Han Q, et al. Emerging concepts and therapeutic implications of beta-adrenergic receptor subtype signaling. Pharmacol Ther 2005; 108(3):257-68.
27 刘超,刘少华,王丽丽,等.β肾上腺素受体住婴幼儿血管瘤发病机制中作用的探讨.口腔医学2011;31(7)：434-5.
28 张凌,麦华明,郑家伟.普萘洛尔治疗婴幼儿血管瘤的作用机制研究进展.中国口腔颌面外科杂志2012;1 0(4)：342-6.
29 McConnaughey MM, Sheets KA, Davis J, et al. Differences in beta-adrenergic receptor densities in omental and subcutaneous adipose tissue from obese African American and Caucasian women. Metabolism 2004; 53(2):247-51.
30 Rutledge DR. Are there beta-adrenergic receptor response differences between racial groups? Dicp 1991; 25(7-8):824-34.
31 Cross HR, Murphy E, Koch WJ, et al. Male and female mice overexpressing the beta(2)-adrenergic receptor exhibit differences in ischemia/reperfusion injury:role of nitric oxide. Cardiovasc Res 2002; 53(3):662-71.
32 Chu SH, Sutherland K, Beck J, et al. Sex differences in expression of calcium-handling proteins and beta-adrenergic receptors in rat heart ventricle. Life Sci 2005; 76(23): 2735-49.
33 Kim YR, Min SK and Yu BH. Differences in beta-adrenergic receptor sensitivity between women and men with panic disorder. Eur Neuropsychopharmacol 2004; 14(6):515-20.
34 Nishiura T, Gao C, Nan X, et al. Expression and postnatal changes of adrenergic receptor subtype mRNAin rat submandibular glands. Arch Oral Biol 2001; 46(7):573-84.
35 Xu C, Liu A, Sun H, et al. beta2-Adrenoceptor confers cardioprotection against hypoxia in isolated ventricular myocytes and the effects depend on estrogenic environment. J Recept Signal Transduct Res 2010; 30(4):255-61.
36 Kaliner M. Mechanisms of glucocorticosteroid action in bronchial asthma. J Allergy Clin Immunol 1985; 76(2 Pt 2):321-9.
37 Campbell S, Park JH, Rowe J, et al. Chorionic villus sampling as a source of trophoblasts. Placenta 2007; 28(11-12):1118-22.
38 Kaplan P, Normandin J, Jr., Wilson GN, et al. Malformations and minor anomalies in children whose mothers had prenatal diagnosis:comparison between CVS and amniocentesis. Am J Med Genet 1990; 37(3):366-70.
39 Burton BK, Schulz CJ, Angle B, et al. An increased incidence of haemangiomas in infants born following chorionic villus sampling (CVS). Prenat Diagn 1995; 15(3):209-14.
40 Sun ZY, Yi CG, Zhao H, et al. Infantile hemangioma is originated from placental trophoblast, fact or fiction? Med Hypotheses 2008; 71(3):444-8.
41 North PE, Waner M, Mizeracki A, et al. GLUT1:a newly discovered immunohistochemical marker for juvenile hemangiomas. Hum Pathol 2000; 31(1):11-22.
42 Pittman KM, Losken HW, Kleinman ME, et al. No evidence for maternal-fetal microchimerism in infantile hemangioma:a molecular genetic investigation. J Invest Dermatol 2006; 126(11):2533-8.
43 Regnier S, Dupin N, Le Danff C, et al. Endothelial cells in infantile haemangiomas originate from the child and not from the mother (a fluorescence in situ hybridization-based study). Br J Dermatol 2007; 157(1):158-60.
44 North PE, Waner M and Brodsky MC. Are infantile hemangiomas of placental origin? Ophthalmology 2002; 109(4):633-4.
45 Li F, Tiede B, Massague J, et al. Beyond tumorigenesis:cancer stem cells in metastasis. Cell Res 2007; 17(1):3-14.
46 Walter JW, North PE, Waner M, et al. Somatic mutation of vascular endothelial growth factor receptors in juvenile hemangioma. Genes Chromosomes Cancer 2002; 33(3): 295-303.
47 Khan ZA, Melero-Martin JM, Wu X, et al. Endothelial progenitor cells from infantile hemangioma and umbilical cord blood display unique cellular responses to endostatin. Blood 2006; 108(3):915-21.
48 Yu Y, Flint AF, Mulliken JB, et al. Endothelial progenitor cells in infantile hemangioma. Blood 2004; 103(4):1373-5.
49 Barnes CM, Christison-Lagay EA and Folkman J. The placenta theory and the origin of infantile hemangioma. Lymphat Res Biol 2007; 5(4):245-55.
50 Dadras SS, North PE, Bertoncini J, et al. Infantile hemangiomas are arrested in an early developmental vascular differentiation state. Mod Pathol 2004; 17(9):1068-79.
51 Yang J, Aguila JR, Alipio Z, et al. Enhanced self-renewal of hematopoietic stem/progenitor cells mediated by the stem cell gene Sall4. J Hematol Oncol 2011; 4:38.
52 Waner M, North PE, Scherer KA, et al. The nonrandom distribution of facial hemangiomas. Arch Dermatol 2003; 139(7):869-75.
53 Chang El, Chang El, Thangarajah H, et al. Hypoxia, hormones, and endothelial progenitor cells in hemangioma. Lymphat Res Biol 2007; 5(4):237-43.
54 Walter JW, Blei F, Anderson JL, et al. Genetic mapping of a novel familial form of infantile hemangioma. Am J Med Genet 1999; 82(1):77-83.
55 Blei F, Walter J, Orlow SJ, et al. Familial segregation of hemangiomas and vascular malformations as an autosomal dominant trait. Arch Dermatol 1998; 134(6):718-22.
56 Chang J, Most D, Bresnick S, et al. Proliferative hemangiomas:analysis of cytokine gene expression and angiogenesis. Plast Reconstr Surg 1999; 103(1):1-9; discussion 10.
57 Sasaki GH, Pang CY and Wittliff JL. Pathogenesis and treatment of infant skin strawberry hemangiomas:clinical and in vitro studies of hormonal effects. Plast Reconstr Surg 1984; 73(3):359-70.
58 Bielenberg DR, Bucana CD, Sanchez R, et al. Progressive growth of infantile cutaneous hemangiomas is directly correlated with hyperplasia and angiogenesis of adjacent epidermis and inversely correlated with expression of the endogenous angiogenesis inhibitor, IFN-beta. Int J Oncol 1999; 14(3):401-8.
59 Takahashi K, Mulliken JB, Kozakewich HP, et al. Cellular markers that distinguish the phases of hemangioma during infancy and childhood. J Clin Invest 1994; 93(6):2357-64.
60 Qu Z, Liebler JM, Powers MR, et al. Mast cells are a major source of basic fibroblast growth factor in chronic inflammation and cutaneous hemangioma. Am J Pathol 1995; 147(3):564-73.
61 Giatromanolaki A, Arvanitidou V, Hatzimichael A, et al. The HIF-2alpha/VEGF pathway activation in cutaneous capillary haemangiomas. Pathology 2005; 37(2):149-51.
62 Kleinman ME, Greives MR, Churgin SS, et al. Hypoxia-induced mediators of stem/progenitor cell trafficking are increased in children with hemangioma. Arterioscler Thromb Vase Bid 2007; 27(12):2664-70.
63 Sun ZY, Yang L, Yi CG, et al. Possibilities and potential roles of estrogen in the pathogenesis of proliferation hemangiomas formation. Med Hypotheses 2008; 71(2): 286-92.
64 Starkey E and Shahidullah H. Propranolol for infantile haemangiomas:a review. Arch Dis Child 2011.
65 Emilien G and Maloteaux JM. Current therapeutic uses and potential of beta-adrenoceptor agonists and antagonists. Eur J Clin Pharmacol 1998; 53(6):389-404.
66 Benish M, Bartal I, Goldfarb Y, et al. Perioperative use of beta-blockers and COX-2 inhibitors may improve immune competence and reduce the risk of tumor metastasis. Ann Surg Oncol 2008; 15(7):2042-52.
67 Park PG, Merryman J, Orloff M, et al. Beta-adrenergic mitogenic signal transduction in peripheral lung adenocarcinoma:implications for individuals with preexisting chronic lung disease. Cancer Res 1995; 55(16):3504-8.
68 Wax PM, Erdman AR, Chyka PA, et al. beta-blocker ingestion:an evidence-based consensus guideline for out-of-hospitai management. Clin Toxicol (Phila) 2005; 43(3): 131-46.
69 Lawley LP, Siegfried E and Todd JL. Propranolol treatment for hemangioma of infancy: risks and recommendations. Pediatr Dermatol 2009; 26(5):610-4.
70 Haider KM, Plager DA, Neely DE, et al. Outpatient treatment of periocular infantile hemangiomas with oral propranolol. J AAPOS 2010; 14(3):251-6.
71 Kuperman S and Stewart MA. Use of propranolol to decrease aggressive outbursts in younger patients. Open study reveals potentially favorable outcome. Psychosomatics 1987; 28(6):315-9.
72 秦中平，刘学建,李克雷,等.小剂最普荼诺尔口服治疗婴幼儿血管瘤的近期疗效与安全性评价.中华医学杂志2009；89(44)：3130-4.
73 Manunza F, Syed S, Laguda B, et al. Propranolol for complicated infantile haemangiomas: a case series of 30 infants. Br J Dermatol 2010; 162(2):466-8.
74 Love JN, Howell JM, Klein-Schwartz W, et al. Lack of toxicity from pediatric beta-blocker exposures. Hum Exp Toxicol 2006; 25(6):341-6.
75 Love JN and Sikka N. Are 1-2 tablets dangerous? Beta-blocker exposure in toddlers. J Emerg Med 2004; 26(3):309-14.
76 Holland KE, Frieden IJ, Frommelt PC, et al. Hypoglycemia in children taking propranolol for the treatment of infantile hemangioma. Arch Dermatol 2010; 146(7):775-8.
77 Abbott J, Parulekar M, Shahidullah H, et al. Diarrhea associated with propranolol treatment for hemangioma of infancy (HOI). Pediatr Dermatol 2010; 27(5):558.
78 Belson MG, Sullivan K and Geller RJ. Beta-adrenergic antagonist exposures in children. Vet Hum Toxicol 2001; 43(6):361-5.
79 Breur JM, de Graaf M, Breugem CC, et al. Hypoglycemia as a result of propranolol during treatment of infantile hemangioma:a case report. Pediatr Dermatol 2011; 28(2):169-71.
80 Kallen RJ, Mohler JH and Lin HL. Hypoglycemia:a complication of treatment of hypertension with propranolol. Clin Pediatr (Phila) 1980; 19(8):567-8.
81 Fraley DS, Bruns FJ, Segel DP, et al. Propranolol-related bronchospasm in patients without history of asthma. South Med J 1980; 73(2):238-40.
82 Guimaraes S and Moura D. Vascular adrenoceptors:an update. Pharmacol Rev 2001; 53(2):319-56.
83 Figueroa XF, Poblete I, Fernandez R, et al. NO production and eNOS phosphorylation induced by epinephrine through the activation of beta-adrenoceptors. Am J Physiol Heart Circ Physiol 2009; 297(1):H134-43.
84 Chen J, Huang C, Zhang B, et al. The effects of carvedilol on cardiac structural remodeling:The role of endogenous nitric oxide in the activity of carvedilol. Mol Med Rep 2013; 7(4):1155-8.
85 Berard M, Sordello S, Ortega N, et al. Vascular endothelial growth factor confers a growth advantage in vitro and in vivo to stromal cells cultured from neonatal hemangiomas. Am J Pathol 1997; 150(4):1315-26.
86 Przewratil P, Sitkiewicz A, Wyka K, et al. Serum levels of vascular endothelial growth factor and basic fibroblastic growth factor in children with hemangiomas and vascular malformations-preliminary report. Pediatr Dermatol 2009; 26(4):399-404.
87 Przewratil P, Sitkiewicz A and Andrzejewska E. Local serum levels of vascular endothelial growth factor in infantile hemangioma:intriguing mechanism of endothelial growth. Cytokine 2010; 49(2):141-7.
88 Lutgendorf SK, Cole S, Costanzo E, et al. Stress-related mediators stimulate vascular endothelial growth factor secretion by two ovarian cancer cell lines. Clin Cancer Res 2003; 9(12):4514-21.
89 Thaker PH, Han LY, Kamat AA, et al. Chronic stress promotes tumor growth and angiogenesis in a mouse model of ovarian carcinoma. Nat Med 2006; 12(8):939-44.
90 Yang EV, Sood AK, Chen M, et al. Norepinephrine up-regulates the expression of vascular endothelial growth factor, matrix metalloproteinase (MMP)-2, and MMP-9 in nasopharyngeal carcinoma tumor cells. Cancer Res 2006; 66(21):10357-64.
91 Annabi B, Lachambre MP, Plouffe K, et al. Propranolol adrenergic blockade inhibits human brain endothelial cells tubulogenesis and matrix metalloproteinase-9 secretion. Pharmacol Res 2009; 60(5):438-45.
92 Parenti A, Morbidelli L, Cui XL, et al. Nitric oxide is an upstream signal of vascular endothelial growth factor-induced extracellular signal-regulated kinase1/2 activation in postcapillary endothelium. J Biol Chem 1998; 273(7):4220-6.
93 Al-Wadei HA, Al-Wadei MH and Schuller HM. Prevention of pancreatic cancer by the beta-blocker propranolol. Anticancer Drugs 2009; 20(6):477-82.
94 Mancini AJ and Smoller BR. Proliferation and apoptosis within juvenile capillary hemangiomas. Am J Dermatopathol 1996; 18(5):505-14.
95 Razon MJ, Kraling BM, Mulliken JB, et al. Increased apoptosis coincides with onset of involution in infantile hemangioma. Microcirculation 1998; 5(2-3):189-95.
96 Sommers Smith SK and Smith DM. Beta blockade induces apoptosis in cultured capillary endothelial cells. In Vitro Cell Dev Biol Anim 2002; 38(5):298-304.
97 Palm D, Lang K, Niggemann B, et al. The norepinephrine-driven metastasis development of PC-3 human prostate cancer cells in BALB/c nude mice is inhibited by beta-blockers. Int J Cancer 2006; 118(11):2744-9.
98 Ganem D. KSHV and the pathogenesis of Kaposi sarcoma:listening to human biology and medicine. J Clin Invest 2010; 120(4):939-49.
99 Hermans DJ, van Beynum IM, van der Vijver RJ, et al. Kaposiform hemangioendothelioma with Kasabach-Merritt syndrome:a new indication for propranolol treatment. J Pediatr Hematol Oncol 2011; 33(4):e171-3.
100 Moss HB, Sines DT, Blatt J, et al. Epithelioid hemangioma responsive to oral propranolol. Ophthal Plast Reconstr Surg 2012; 28(4):e88-90.
2 Bivings L. Spontaneous regression of angiomas in children; twenty-two years' observation covering 236 cases. J Pediatr 1954; 45(6):643-7.
3 Jacobs AH. Strawberry hemangiomas; the natural history of the untreated lesion. Calif Med 1957; 86(1):8-10.
4 郑家伟,杨秀娟.血管瘤的治疗选择.中国实用口腔科杂志 2009：2(5)：274-9.
5 Enjolras O and Mulliken JB. The current management of vascular birthmarks. Pediatr Dermatol 1993; 10(4):311-3.
6 Margileth AM and Museles M. Cutaneous hemangiomas in children. Diagnosis and conservative management. Jama 1965; 194(5):523-6.
7 Robertson RL, Robson CD, Barnes PD, et al. Head and neck vascular anomalies of childhood. Neuroimaging Clin NAm 1999; 9(1):115-32.
8 Rutledge DR. Are there beta-adrenergic receptor response differences between racial groups? Dicp 1991; 25(7-8):824-34.
9 Holland KE and Drolet BA. Infantile hemangioma. Pediatr Clin North Am 2010; 57(5): 1069-83.
10 Jia J and Zhao YF. Biomarkers:important clues to the pathogenesis of infantile haemangioma and their clinical significance. Chin J Dent Res 2010; 13(2):105-8.
11 Ritter EJ, Goldman L, Richfield D, et al. The chicken comb and wattle as experimental model for investigative argon laser therapy of angiomas. Acta Derm Venereol 1969; 49(3): 304-8.
12 Sato N, Sato T, Takahashi S, et al. Establishment of murine endothelial cell lines that develop angiosarcomas in vivo:brief demonstration of a proposed animal model for Kaposi's sarcoma. Cancer Res 1986; 46(1):362-6.
13 Bautch VL, Toda S, Hassell JA, et al. Endothelial cell tumors develop in transgenic mice carrying polyoma virus middle Toncogene. Cell 1987; 51(4):529-37.
14 虎小毅,杨壮群,宋勇吗,等.细胞悬液接种法血管瘤裸鼠动物模型的建立.中国美容医学2008；17(7)：1012-4.
15 Tang Y, Liu W, Yu S, et al. A novel in vivo model of human hemangioma: xenograft of human hemangioma tissue on nude mice. Plast Reconstr Surg 2007; 120(4):869-78.
16 Hohenleutner U, Landthaler M, Hamm H, et al. Hemangiomas of infancy and childhood. J Dtsch Dermatol Ges 2007; 5(4):334-8.
17 Akhavan A and Zippin JH. Current treatments for infantile hemangiomas. J Drugs Dermatol 2010; 9(2):176-80.
18 Berard M, Sordello S, Ortega N, et al. Vascular endothelial growth factor confers a growth advantage in vitro and in vivo to stromal cells cultured from neonatal hemangiomas. Am J Pathol 1997; 150(4):1315-26.
19 Chang J, Most D, Bresnick S, et al. Proliferative hemangiomas:analysis of cytokine gene expression and angiogenesis. Plast Reconstr Surg 1999; 103(1):1-9; discussion 10.
20 Giatromanolaki A, Arvanitidou V, Hatzimichael A, et al. The HIF-2alpha/VEGF pathway activation in cutaneous capillary haemangiomas. Pathology 2005; 37(2):149-51.
21 Kleinman ME, Greives MR, Churgin SS, et al. Hypoxia-induced mediators of stem/progenitor cell trafficking are increased in children with hemangioma. Arterioscler Thromb Vase Biol 2007; 27(12):2664-70.
22 Qu Z, Liebler JM, Powers MR, et al. Mast cells are a major source of basic fibroblast growth factor in chronic inflammation and cutaneous hemangioma. Am J Pathol 1995; 147(3):564-73.
23 Takahashi K, Mulliken JB, Kozakewich HP, et al. Cellular markers that distinguish the phases of hemangioma during infancy and childhood. J Clin Invest 1994; 93(6):2357-64.
24 Mulliken JB, Fishman SJ and Burrows PE. Vascular anomalies. Curr Probl Surg 2000; 37(8):517-84.
25 Schwartz RA, Sidor Ml, Musumeci ML, et al. Infantile haemangiomas:a challenge in paediatric dermatology. J Eur Acad Dermatol Venereol 2010; 24(6):631-8.
26 Soldatskii lu L, Shekhter AB, Ponkratenko AD, et al. [Effect of cryotherapy and laser destruction on experimental model of human vascular tumor]. Vestn Otorinolaringol 1995(2):10-4.
27 Anvari B, Tanenbaum BS, Hoffman W, et al. Nd:YAG laser irradiation in conjunction with cryogen spray cooling induces deep and spatially selective photocoagulation in animal models. Phys Med Biol 1997; 42(2):265-82.
28 Softer D, Resnick-Roguel N, Eldor A, et al. Multifocal vascular tumors in fowl induced by a newly isolated retrovirus. Cancer Res 1990; 50(15):4787-93.
29 Liekens S, Andrei G, Vandeputte M, et al. Potent inhibition of hemangioma formation in rats by the acyclic nucleoside phosphonate analogue cidofovir. Cancer Res 1998; 58(12): 2562-7.
30 Taraboletti G, Belotti D, Dejana E, et al. Endothelial cell migration and invasiveness are induced by a soluble factor produced by murine endothelioma cells transformed by polyoma virus middle T oncogene. Cancer Res 1993; 53(16):3812-6.
31 Kiefer F, Anhauser I, Soriano P, et al. Endothelial cell transformation by polyomavirus middle T antigen in mice lacking Src-related kinases. Curr Biol 1994; 4(2):100-9.
32 Lee RJ, Springer ML, Blanco-Bose WE, et al. VEGF gene delivery to myocardium: deleterious effects of unregulated expression. Circulation 2000; 102(8):898-901.
33 Flanagan SP.'Nude', a new hairless gene with pleiotropic effects in the mouse. Genet Res 1966; 8(3):295-309.
34 Povlsen CO. Heterotransplants of human tumors in nude mice. Antibiot Chemother 1980; 28:15-20.
35 Sharkey FE and Fogh J. Considerations in the use of nude mice for cancer research. Cancer Metastasis Rev 1984; 3(4):341-60.
36 van Weerden WM and Romijn JC. Use of nude mouse xenograft models in prostate cancer research. Prostate 2000; 43(4):263-71.
37 Valerio MG, Fineman EL, Bowman RL, et al. Long-term survival of normal adult human tissues as xenografts in congenially athymic nude mice. J Natl Cancer Inst 1981; 66(5): 849-58.
38 Kischer CW, Sheridan D and Pindur J. Use of nude (athymic) mice for the study of hypertrophic scars and keloids:vascular continuity between mouse and implants. Anat Rec 1989; 225(3):189-96.
39 Gilhar A and Etzioni A. The nude mouse model for the study of human skin disorders. Dermatology 1994; 189(1):5-8.
40 Gerber H, Wagner HE, Burgi U, et al. Model of the athymic nude mouse for the study of benign goiter disease. Exp Clin Endocrinol Diabetes 1996; 104 Suppl 3:56-9.
41 Pouliot R, Larouche D, Auger FA, et al. Reconstructed human skin produced in vitro and grafted on athymic mice. Transplantation 2002; 73(11):1751-7.
42 Honaramooz A, Snedaker A, Boiani M, et al. Sperm from neonatal mammalian testes grafted in mice. Nature 2002; 418(6899):778-81.
43 Guerret S, Govignon E, Hartmann DJ, et al. Long-term remodeling of a bilayered living human skin equivalent (Apligraf) grafted onto nude mice:immunolocalization of human cells and characterization of extracellular matrix. Wound Repair Regen 2003; 11(1): 35-45.
44 Martin-Padura I, De Castellarnau C, Uccini S, et al. Expression of VE (vascular endothelial)-cadherin and other endothelial-specific markers in haemangiomas. J Pathol 1995; 175(1):51-7.
45 Dosanjh A, Chang J, Bresnick S, et al. In vitro characteristics of neonatal hemangioma endothelial cells:similarities and differences between normal neonatal and fetal endothelial cells. J Cutan Pathol 2000; 27(9):441-50.
46赵曜,谭玉珍,周良辅,等.中枢神经系统海绵状血管瘤内皮细胞的分离和培养.中华实验外科杂志2003;20(8)：685-6
47 Gould GW and Holman GD. The glucose transporter family:structure, function and tissue-specific expression. Biochem J 1993; 295 (Pt2):329-41.
48 Jansson T, Wennergren M and Powell TL Placental glucose transport and GLUT 1 expression in insulin-dependent diabetes. Am J Obstet Gynecol 1999; 180(1 Pt 1):163-8.
49 Schlingemann RO, Hofman P, Klooster J, et al. Ciliary muscle capillaries have blood-tissue barrier characteristics. Exp Eye Res 1998; 66(6):747-54.
50 North PE, Waner M, Mizeracki A, et al. GLUT1:a newly discovered immunohistochemical marker for juvenile hemangiomas. Hum Pathol 2000; 31(1):11-22.
51 Mo JQ, Dimashkieh HH and Bove KE. GLUT1 endothelial reactivity distinguishes hepatic infantile hemangioma from congenital hepatic vascular malformation with associated capillary proliferation. Hum Pathol 2004; 35(2):200-9.
52 Lo K, Mihm M and Fay A. Current theories on the pathogenesis of infantile hemangioma. Semin Ophthalmol 2009; 24(3):172-7.
53 Barnes CM, Huang S, Kaipainen A, et al. Evidence by molecular profiling for a placental origin of infantile hemangioma. Proc Natl Acad Sci U S A 2005; 102(52):19097-102.
54 North PE, Waner M, Mizeracki A, et al. A unique microvascular phenotype shared by juvenile hemangiomas and human placenta. Arch Dermatoi 2001; 137(5):559-70.
55 Khan ZA, Boscolo E, Picard A, et al. Multipotential stem cells recapitulate human infantile hemangioma in immunodeficient mice. J Clin Invest 2008; 118(7):2592-9.
1 laccarino G, Ciccarelli M, Sorriento D, et al. Ischemic neoangiogenesis enhanced by beta2-adrenergic receptor overexpression:a novel role for the endothelial adrenergic system. Circ Res 2005; 97(11):1182-9.
2 Chisholm KM, Chang KW, Truong MT, et al. beta-Adrenergic receptor expression in vascular tumors. Mod Pathol 2012.
3 Fredriksson JM, Lindquist JM, Bronnikov GE, et al. Norepinephrine induces vascular endothelial growth factor gene expression in brown adipocytes through a beta-adrenoreceptor/cAMP/protein kinase A pathway involving Src but independently of Erk1/2. J Biol Chem 2000; 275(18):13802-11.
4 Zhang D, Ma Q, Shen S, et al. Inhibition of pancreatic cancer cell proliferation by propranolol occurs through apoptosis induction:the study of beta-adrenoceptor antagonist's anticancer effect in pancreatic cancer cell. Pancreas 2009; 38(1):94-100.
5 Jia J and Zhao YF. Biomarkers:important clues to the pathogenesis of infantile haemangioma and their clinical significance. Chin J Dent Res 2010; 13(2):105-8.
6 Holland KE and Drolet BA. Infantile hemangioma. Pediatr Clin North Am 2010; 57(5): 1069-83.
7 郑家伟,杨秀娟.血管瘤的治疗选择.中国实用口腔科杂志2009;2(5)：274-9.
8 Mulliken JB, Fishman SJ and Burrows PE. Vascular anomalies. Curr Probl Surg 2000; 37(8):517-84.
9 Waner M and Suen JY. Management of congenital vascular lesions of the head and neck. Oncology (Williston Park) 1995; 9(10):989-94,97; discussion 98 passim.
10 Mulliken JB and Glowacki J. Classification of pediatric vascular lesions. Plast Reconstr Surg 1982; 70(1):120-1.
11 Hohenleutner U, Landthaler M, Hamm H, et al. Hemangiomas of infancy and childhood. J Dtsch Dermatol Ges 2007; 5(4):334-8.
12 Storch CH and Hoeger PH. Propranolol for infantile haemangiomas:insights into the molecular mechanisms of action. Br J Dermatol 2010; 163(2):269-74.
13 Akhavan A and Zippin JH. Current treatments for infantile hemangiomas. J Drugs Dermatol 2010; 9(2):176-80.
14仇雅璟,马刚,林晓曦.婴幼儿血管瘤发生与形成机制的研究进展.中华整形外科杂志2012;28(3)：233-6.
15 Boye E, Yu Y, Paranya G, et al. Clonality and altered behavior of endothelial cells from hemangiomas. J Clin Invest 2001; 107(6):745-52.
16 Barnes CM, Huang S, Kaipainen A, et al. Evidence by molecular profiling for a placental origin of infantile hemangioma. Proc Natl Acad Sci U S A 2005; 102(52):19097-102.
17 Khan ZA, Boscolo E, Picard A, et al. Multipotential stem cells recapitulate human infantile hemangioma in immunodeficient mice. J Clin Invest 2008; 118(7):2592-9.
18 North PE, Waner M, Mizeracki A, et al. A unique microvascular phenotype shared by juvenile hemangiomas and human placenta. Arch Dermatol 2001; 137(5):559-70.
19 Leaute-Labreze C, Dumas de la Roque E, Hubiche T, et al. Propranolol for severe hemangiomas of infancy. N Engl J Med 2008; 358(24):2649-51.
20 Buckmiller LM, Munson PD, Dyamenahalli U, et al. Propranolol for infantile hemangiomas: early experience at a tertiary vascular anomalies center. Laryngoscope 2010; 120(4): 676-81.
21 Sans V, Dumas de la Roque E, Berge J, et al. Propranolol for Severe Infantile Hemangiomas:Follow-Up Report. Pediatrics 2009.
22 Denoyelle F, Leboulanger N, Enjolras O, et al. Role of Propranolol in the therapeutic strategy of infantile laryngotracheal hemangioma. Int J Pediatr Otorhinolaryngol 2009; 73(8):1168-72.
23 Rosbe KW, Suh KY, Meyer AK, et al. Propranolol in the management of airway infantile hemangiomas. Arch Otolaryngol Head Neck Surg 2010; 136(7):658-65.
24 Marsciani A, Pericoli R, Alaggio R, et al. Massive response of severe infantile hepatic hemangioma to propanolol. Pediatr Blood Cancer 2010; 54(1):176.
25 Li YC, McCahon E, Rowe NA, et al. Successful treatment of infantile haemangiomas of the orbit with propranolol. Clin Experiment Ophthalmol 2010; 38(6):554-9.
26 Zheng M, Zhu W, Han Q, et al. Emerging concepts and therapeutic implications of beta-adrenergic receptor subtype signaling. Pharmacol Ther 2005; 108(3):257-68.
27 刘超,刘少华,王丽丽,等.β肾上腺素受体住婴幼儿血管瘤发病机制中作用的探讨.口腔医学2011;31(7)：434-5.
28 张凌,麦华明,郑家伟.普萘洛尔治疗婴幼儿血管瘤的作用机制研究进展.中国口腔颌面外科杂志2012;1 0(4)：342-6.
29 McConnaughey MM, Sheets KA, Davis J, et al. Differences in beta-adrenergic receptor densities in omental and subcutaneous adipose tissue from obese African American and Caucasian women. Metabolism 2004; 53(2):247-51.
30 Rutledge DR. Are there beta-adrenergic receptor response differences between racial groups? Dicp 1991; 25(7-8):824-34.
31 Cross HR, Murphy E, Koch WJ, et al. Male and female mice overexpressing the beta(2)-adrenergic receptor exhibit differences in ischemia/reperfusion injury:role of nitric oxide. Cardiovasc Res 2002; 53(3):662-71.
32 Chu SH, Sutherland K, Beck J, et al. Sex differences in expression of calcium-handling proteins and beta-adrenergic receptors in rat heart ventricle. Life Sci 2005; 76(23): 2735-49.
33 Kim YR, Min SK and Yu BH. Differences in beta-adrenergic receptor sensitivity between women and men with panic disorder. Eur Neuropsychopharmacol 2004; 14(6):515-20.
34 Nishiura T, Gao C, Nan X, et al. Expression and postnatal changes of adrenergic receptor subtype mRNAin rat submandibular glands. Arch Oral Biol 2001; 46(7):573-84.
35 Xu C, Liu A, Sun H, et al. beta2-Adrenoceptor confers cardioprotection against hypoxia in isolated ventricular myocytes and the effects depend on estrogenic environment. J Recept Signal Transduct Res 2010; 30(4):255-61.
36 Kaliner M. Mechanisms of glucocorticosteroid action in bronchial asthma. J Allergy Clin Immunol 1985; 76(2 Pt 2):321-9.
37 Campbell S, Park JH, Rowe J, et al. Chorionic villus sampling as a source of trophoblasts. Placenta 2007; 28(11-12):1118-22.
38 Kaplan P, Normandin J, Jr., Wilson GN, et al. Malformations and minor anomalies in children whose mothers had prenatal diagnosis:comparison between CVS and amniocentesis. Am J Med Genet 1990; 37(3):366-70.
39 Burton BK, Schulz CJ, Angle B, et al. An increased incidence of haemangiomas in infants born following chorionic villus sampling (CVS). Prenat Diagn 1995; 15(3):209-14.
40 Sun ZY, Yi CG, Zhao H, et al. Infantile hemangioma is originated from placental trophoblast, fact or fiction? Med Hypotheses 2008; 71(3):444-8.
41 North PE, Waner M, Mizeracki A, et al. GLUT1:a newly discovered immunohistochemical marker for juvenile hemangiomas. Hum Pathol 2000; 31(1):11-22.
42 Pittman KM, Losken HW, Kleinman ME, et al. No evidence for maternal-fetal microchimerism in infantile hemangioma:a molecular genetic investigation. J Invest Dermatol 2006; 126(11):2533-8.
43 Regnier S, Dupin N, Le Danff C, et al. Endothelial cells in infantile haemangiomas originate from the child and not from the mother (a fluorescence in situ hybridization-based study). Br J Dermatol 2007; 157(1):158-60.
44 North PE, Waner M and Brodsky MC. Are infantile hemangiomas of placental origin? Ophthalmology 2002; 109(4):633-4.
45 Li F, Tiede B, Massague J, et al. Beyond tumorigenesis:cancer stem cells in metastasis. Cell Res 2007; 17(1):3-14.
46 Walter JW, North PE, Waner M, et al. Somatic mutation of vascular endothelial growth factor receptors in juvenile hemangioma. Genes Chromosomes Cancer 2002; 33(3): 295-303.
47 Khan ZA, Melero-Martin JM, Wu X, et al. Endothelial progenitor cells from infantile hemangioma and umbilical cord blood display unique cellular responses to endostatin. Blood 2006; 108(3):915-21.
48 Yu Y, Flint AF, Mulliken JB, et al. Endothelial progenitor cells in infantile hemangioma. Blood 2004; 103(4):1373-5.
49 Barnes CM, Christison-Lagay EA and Folkman J. The placenta theory and the origin of infantile hemangioma. Lymphat Res Biol 2007; 5(4):245-55.
50 Dadras SS, North PE, Bertoncini J, et al. Infantile hemangiomas are arrested in an early developmental vascular differentiation state. Mod Pathol 2004; 17(9):1068-79.
51 Yang J, Aguila JR, Alipio Z, et al. Enhanced self-renewal of hematopoietic stem/progenitor cells mediated by the stem cell gene Sall4. J Hematol Oncol 2011; 4:38.
52 Waner M, North PE, Scherer KA, et al. The nonrandom distribution of facial hemangiomas. Arch Dermatol 2003; 139(7):869-75.
53 Chang El, Chang El, Thangarajah H, et al. Hypoxia, hormones, and endothelial progenitor cells in hemangioma. Lymphat Res Biol 2007; 5(4):237-43.
54 Walter JW, Blei F, Anderson JL, et al. Genetic mapping of a novel familial form of infantile hemangioma. Am J Med Genet 1999; 82(1):77-83.
55 Blei F, Walter J, Orlow SJ, et al. Familial segregation of hemangiomas and vascular malformations as an autosomal dominant trait. Arch Dermatol 1998; 134(6):718-22.
56 Chang J, Most D, Bresnick S, et al. Proliferative hemangiomas:analysis of cytokine gene expression and angiogenesis. Plast Reconstr Surg 1999; 103(1):1-9; discussion 10.
57 Sasaki GH, Pang CY and Wittliff JL. Pathogenesis and treatment of infant skin strawberry hemangiomas:clinical and in vitro studies of hormonal effects. Plast Reconstr Surg 1984; 73(3):359-70.
58 Bielenberg DR, Bucana CD, Sanchez R, et al. Progressive growth of infantile cutaneous hemangiomas is directly correlated with hyperplasia and angiogenesis of adjacent epidermis and inversely correlated with expression of the endogenous angiogenesis inhibitor, IFN-beta. Int J Oncol 1999; 14(3):401-8.
59 Takahashi K, Mulliken JB, Kozakewich HP, et al. Cellular markers that distinguish the phases of hemangioma during infancy and childhood. J Clin Invest 1994; 93(6):2357-64.
60 Qu Z, Liebler JM, Powers MR, et al. Mast cells are a major source of basic fibroblast growth factor in chronic inflammation and cutaneous hemangioma. Am J Pathol 1995; 147(3):564-73.
61 Giatromanolaki A, Arvanitidou V, Hatzimichael A, et al. The HIF-2alpha/VEGF pathway activation in cutaneous capillary haemangiomas. Pathology 2005; 37(2):149-51.
62 Kleinman ME, Greives MR, Churgin SS, et al. Hypoxia-induced mediators of stem/progenitor cell trafficking are increased in children with hemangioma. Arterioscler Thromb Vase Bid 2007; 27(12):2664-70.
63 Sun ZY, Yang L, Yi CG, et al. Possibilities and potential roles of estrogen in the pathogenesis of proliferation hemangiomas formation. Med Hypotheses 2008; 71(2): 286-92.
64 Starkey E and Shahidullah H. Propranolol for infantile haemangiomas:a review. Arch Dis Child 2011.
65 Emilien G and Maloteaux JM. Current therapeutic uses and potential of beta-adrenoceptor agonists and antagonists. Eur J Clin Pharmacol 1998; 53(6):389-404.
66 Benish M, Bartal I, Goldfarb Y, et al. Perioperative use of beta-blockers and COX-2 inhibitors may improve immune competence and reduce the risk of tumor metastasis. Ann Surg Oncol 2008; 15(7):2042-52.
67 Park PG, Merryman J, Orloff M, et al. Beta-adrenergic mitogenic signal transduction in peripheral lung adenocarcinoma:implications for individuals with preexisting chronic lung disease. Cancer Res 1995; 55(16):3504-8.
68 Wax PM, Erdman AR, Chyka PA, et al. beta-blocker ingestion:an evidence-based consensus guideline for out-of-hospitai management. Clin Toxicol (Phila) 2005; 43(3): 131-46.
69 Lawley LP, Siegfried E and Todd JL. Propranolol treatment for hemangioma of infancy: risks and recommendations. Pediatr Dermatol 2009; 26(5):610-4.
70 Haider KM, Plager DA, Neely DE, et al. Outpatient treatment of periocular infantile hemangiomas with oral propranolol. J AAPOS 2010; 14(3):251-6.
71 Kuperman S and Stewart MA. Use of propranolol to decrease aggressive outbursts in younger patients. Open study reveals potentially favorable outcome. Psychosomatics 1987; 28(6):315-9.
72 秦中平，刘学建,李克雷,等.小剂最普荼诺尔口服治疗婴幼儿血管瘤的近期疗效与安全性评价.中华医学杂志2009；89(44)：3130-4.
73 Manunza F, Syed S, Laguda B, et al. Propranolol for complicated infantile haemangiomas: a case series of 30 infants. Br J Dermatol 2010; 162(2):466-8.
74 Love JN, Howell JM, Klein-Schwartz W, et al. Lack of toxicity from pediatric beta-blocker exposures. Hum Exp Toxicol 2006; 25(6):341-6.
75 Love JN and Sikka N. Are 1-2 tablets dangerous? Beta-blocker exposure in toddlers. J Emerg Med 2004; 26(3):309-14.
76 Holland KE, Frieden IJ, Frommelt PC, et al. Hypoglycemia in children taking propranolol for the treatment of infantile hemangioma. Arch Dermatol 2010; 146(7):775-8.
77 Abbott J, Parulekar M, Shahidullah H, et al. Diarrhea associated with propranolol treatment for hemangioma of infancy (HOI). Pediatr Dermatol 2010; 27(5):558.
78 Belson MG, Sullivan K and Geller RJ. Beta-adrenergic antagonist exposures in children. Vet Hum Toxicol 2001; 43(6):361-5.
79 Breur JM, de Graaf M, Breugem CC, et al. Hypoglycemia as a result of propranolol during treatment of infantile hemangioma:a case report. Pediatr Dermatol 2011; 28(2):169-71.
80 Kallen RJ, Mohler JH and Lin HL. Hypoglycemia:a complication of treatment of hypertension with propranolol. Clin Pediatr (Phila) 1980; 19(8):567-8.
81 Fraley DS, Bruns FJ, Segel DP, et al. Propranolol-related bronchospasm in patients without history of asthma. South Med J 1980; 73(2):238-40.
82 Guimaraes S and Moura D. Vascular adrenoceptors:an update. Pharmacol Rev 2001; 53(2):319-56.
83 Figueroa XF, Poblete I, Fernandez R, et al. NO production and eNOS phosphorylation induced by epinephrine through the activation of beta-adrenoceptors. Am J Physiol Heart Circ Physiol 2009; 297(1):H134-43.
84 Chen J, Huang C, Zhang B, et al. The effects of carvedilol on cardiac structural remodeling:The role of endogenous nitric oxide in the activity of carvedilol. Mol Med Rep 2013; 7(4):1155-8.
85 Berard M, Sordello S, Ortega N, et al. Vascular endothelial growth factor confers a growth advantage in vitro and in vivo to stromal cells cultured from neonatal hemangiomas. Am J Pathol 1997; 150(4):1315-26.
86 Przewratil P, Sitkiewicz A, Wyka K, et al. Serum levels of vascular endothelial growth factor and basic fibroblastic growth factor in children with hemangiomas and vascular malformations-preliminary report. Pediatr Dermatol 2009; 26(4):399-404.
87 Przewratil P, Sitkiewicz A and Andrzejewska E. Local serum levels of vascular endothelial growth factor in infantile hemangioma:intriguing mechanism of endothelial growth. Cytokine 2010; 49(2):141-7.
88 Lutgendorf SK, Cole S, Costanzo E, et al. Stress-related mediators stimulate vascular endothelial growth factor secretion by two ovarian cancer cell lines. Clin Cancer Res 2003; 9(12):4514-21.
89 Thaker PH, Han LY, Kamat AA, et al. Chronic stress promotes tumor growth and angiogenesis in a mouse model of ovarian carcinoma. Nat Med 2006; 12(8):939-44.
90 Yang EV, Sood AK, Chen M, et al. Norepinephrine up-regulates the expression of vascular endothelial growth factor, matrix metalloproteinase (MMP)-2, and MMP-9 in nasopharyngeal carcinoma tumor cells. Cancer Res 2006; 66(21):10357-64.
91 Annabi B, Lachambre MP, Plouffe K, et al. Propranolol adrenergic blockade inhibits human brain endothelial cells tubulogenesis and matrix metalloproteinase-9 secretion. Pharmacol Res 2009; 60(5):438-45.
92 Parenti A, Morbidelli L, Cui XL, et al. Nitric oxide is an upstream signal of vascular endothelial growth factor-induced extracellular signal-regulated kinase1/2 activation in postcapillary endothelium. J Biol Chem 1998; 273(7):4220-6.
93 Al-Wadei HA, Al-Wadei MH and Schuller HM. Prevention of pancreatic cancer by the beta-blocker propranolol. Anticancer Drugs 2009; 20(6):477-82.
94 Mancini AJ and Smoller BR. Proliferation and apoptosis within juvenile capillary hemangiomas. Am J Dermatopathol 1996; 18(5):505-14.
95 Razon MJ, Kraling BM, Mulliken JB, et al. Increased apoptosis coincides with onset of involution in infantile hemangioma. Microcirculation 1998; 5(2-3):189-95.
96 Sommers Smith SK and Smith DM. Beta blockade induces apoptosis in cultured capillary endothelial cells. In Vitro Cell Dev Biol Anim 2002; 38(5):298-304.
97 Palm D, Lang K, Niggemann B, et al. The norepinephrine-driven metastasis development of PC-3 human prostate cancer cells in BALB/c nude mice is inhibited by beta-blockers. Int J Cancer 2006; 118(11):2744-9.
98 Ganem D. KSHV and the pathogenesis of Kaposi sarcoma:listening to human biology and medicine. J Clin Invest 2010; 120(4):939-49.
99 Hermans DJ, van Beynum IM, van der Vijver RJ, et al. Kaposiform hemangioendothelioma with Kasabach-Merritt syndrome:a new indication for propranolol treatment. J Pediatr Hematol Oncol 2011; 33(4):e171-3.
100 Moss HB, Sines DT, Blatt J, et al. Epithelioid hemangioma responsive to oral propranolol. Ophthal Plast Reconstr Surg 2012; 28(4):e88-90.