【作者】 徐广琪；

【导师】 霍然；

【作者基本信息】 山东大学 ， 外科学， 2014， 博士

【摘要】 论文纲要血管瘤(hemangioma)是婴幼儿最常见的先天性良性肿瘤,新生儿发病率2-3%,1岁以下儿童的发病率在10%左右。普萘洛尔作为非选择性β-肾上腺素受体阻滞剂,主要用于治疗心脏疾病如高血压、心绞痛、心动过速等。2008年,法国医生Leaute-Labreze C等偶然发现该药可有效抑制血管瘤的增殖,并加速瘤体消退。之后,国内外众多学者也相续将其应用于血管瘤的治疗,广泛的临床应用证实了该药对血管瘤具有明显疗效。口服普萘洛尔治疗增生期血管瘤疗效显著,同时对消退期血管瘤也有一定效果,目前已逐步取代激素成为治疗婴幼儿血管瘤的一线用药。然而,儿童口服普萘洛尔尚存在很多潜在的副作用,尤其是口服初期,需住院检测心率、血压、血糖等指标,给患儿家长及医师带来诸多不便。能否改变给药途径,采取瘤体局部外涂普萘洛尔制剂的方式,以减轻口服药物所致的潜在不良反应,鉴于此,在第一章第一部分通过对25例门诊“等待观察”的浅表型婴幼儿血管瘤患者初步尝试应用1%普萘洛尔软膏外涂治疗,结果表明疗效显著且未发现可检测到的与外用普萘洛尔软膏相关的不良反应。此外,口服普萘洛尔治疗血管瘤,国内外文献较多集中在对疗效评估,而对服药期间因治疗所致的副作用或不良反应发生情况研究尚存在不足。故在本章第二部分,回顾性分析近年来我科接受口服普萘洛尔治疗的106例血管瘤患儿,以评估药物治疗相关的副作用或不良反应发生情况,为用药安全提供依据。Folkman等学者认为,血管瘤是一种血管形成性质疾病,失控的血管生成是其增殖的主要因素。该过程包括了内皮细胞增殖、细胞外基质的选择性降解、细胞的迁移以及血管通透性的增加。细胞生长因子是一类能诱导或促进新血管形成的物质,可调控内皮细胞的增殖,影响血管形成,对血管瘤的发生发展起关键作用。类表皮生长因子域7(EGFL7)是一种内皮细胞特异性分泌因子,选择性地表达于肿瘤新生血管和增生性组织中,在血管生成过程中起关键作用。由此我们提出,①EGFL7是否在血管瘤组织中表达及在该病发生发展的不同时期是否存在差异表达?②普萘洛尔对血管瘤显著的临床疗效是否通过抑制EGFL7表达?上述问题是进一步研究普萘洛尔对血管瘤作用机制所要解决的问题。鉴于此,第二章第一部分利用免疫组织化学染色技术检测类表皮生长因子域7(EGFL7)在婴幼儿血管瘤组织中的表达,并探讨其在血管瘤发生发展过程中的作用和意义。本章第二部分,通过检测普萘洛尔干预下人脐静脉内皮细胞(HUVEC)增殖、成管、凋亡及EGFL7的表达影响,初步探讨普萘洛尔抑制血管瘤增生的作用机制。第一章普萘洛尔治疗婴幼儿血管瘤临床观察第一部分普萘洛尔外用软膏治疗浅表型婴幼儿血管瘤疗效评估目的探讨局部外涂普萘洛尔软膏治疗浅表型婴幼儿血管瘤的临床疗效及安全性。方法本组共25例(共28处血管瘤,女21例,男4例,年龄1月至10月,平均4个月)浅表型婴幼儿血管瘤患儿,门诊接受瘤体表面外涂1%普萘洛尔软膏,每日3次,均匀涂于瘤体表面,时间5周至59周(平均21周),依据服药前后瘤体表面张力、大小、颜色变化,按照显著有效、部分有效、无效3级评分法对近期疗效进行评价。同时,观察用药后副作用以评估用药安全性。结果28处血管瘤中,16(57%)例显著有效；9(33%)例部分有效；3(10%)例为无效。1%普萘洛尔软膏对浅表型婴幼儿血管瘤的总有效率为90%(95%可信区间72%-98%)。所有患儿均未出现可检测到的不良反应。结论外涂普萘洛尔软膏能有效促进浅表型婴幼儿血管瘤的消退,且未发现口服普萘洛尔常见的不良反应,可以作为婴幼儿血管瘤随访观察期间的一种安全、有效辅助治疗手段。第二部分口服普萘洛尔治疗婴幼儿血管瘤不良反应观察目的：观察口服普萘洛尔治疗婴幼儿血管瘤不良反应,评估其用药安全性。方法：2009年9月至2013年11月间山东省立医院烧伤整形美容科口服普萘洛尔治疗的106例婴幼儿血管瘤患儿,女性71例,男性35例,年龄1-14个月(平均5.1个月)。服药剂量每日1-1.5mg/kg,分3次给药,随访2-10个月,期间对出现的用药相关副作用或并发症进行安全性评估。结果：106例患儿服药期间及随访阶段瘤体均得到有效控制且有程度不同的消退。18例病人(17%,18/106,4例并发两种以上副作用)出现与用药相关的副作用,其中10(9.4%)例口服期间发生腹泻,7(6.6%)例出现一过性血压下降,2(1.9%)例溢奶,2(1.9%)例夜间睡眠差,易激和四肢末端发凉各1例(0.94%)。结论：小剂量(1-1.5mg/kg.day)、分次口服普萘洛尔治疗婴幼儿血管瘤疗效显著,且不良反应发生率低,用药安全,可作为婴治疗幼儿血管瘤的用药选择。第二章普萘洛尔抑制婴幼儿血管瘤增殖的机制探讨第一部分类表皮生长因子域7(EGFL7)在婴幼儿血管瘤组织中的表达及意义目的：检测类表皮生长因子域7(EGFL7)在不同时期婴幼儿血管瘤组织中的表达情况并探讨其在该病发生发展过程中的作用及意义。方法：收集2007年5至2013年9月山东大学附属省立医院烧伤整形美容科手术切除组织病理诊断为婴幼儿血管瘤的石蜡标本49例(男16例,女33例),依据Mulliken标准将标本分成增生期组、退期组、消退完成期组,以正常皮肤作为对照组。应用免疫组织化学法对各期婴幼儿血管瘤组织及正常皮肤中的类表皮生长因子域7(EGFL7)表达情况进行检测,利用计算机图像分析技术分别测量其平均光密度。结果：EGFL7阳性反应定位于血管瘤细胞胞浆中,在血管瘤增生期强阳性或阳性表达,消退期阳性表达,消退完成期和正常皮肤中弱阳性或阴性性表达。结论：EGFL7与增生期婴幼儿血管瘤关系密切,参与了婴幼儿血管瘤病理变化过程,在该病增生期和消退早期细胞增殖和血管形成阶段起重要作用。第二部分普萘洛尔对人脐静脉内皮细胞(HUVEC)生物学行为及EGFL-7表达的影响目的：探讨普萘洛尔对人脐静脉内皮细胞增殖、成管能力、凋亡及EGFL-7表达影响,为进一步研究普萘洛尔治疗婴幼儿血管瘤机制提供理论依据。方法：以离体的人脐静脉内皮细胞为实验对象,采用MTT法、成管实验、流式细胞仪测定不同浓度的普萘洛尔(0μmol/L、25μmo1/L、50μmol/L、100μmo1/L、125μmol/L、150μmol/L、175μmol/L、200μmol/L)对HUVEC增殖、成管能力、凋亡的影响及RT-PCR、western blot法测定类表皮生长因子域-7的变化。结果：普萘洛尔药物干预24和48小时后,药物浓度为≥25um时对人脐静脉内皮细胞增殖能力的抑制率与正常对照组相比有统计学意义(P<0.01),且在25μmol/L、50μmol/L、100μmol/L、125μmol/L浓度下抑制率成明显上升趋势；流式细胞仪检测不同浓度普萘洛尔实验组和空白组对比(24小时),细胞凋亡率逐步增加；药物处理组24h细胞成管个数与阴性对照无统计学差异,48小时对成管能力有一定抑制作用；RT-PCR和Western blot实验结果显示药物处理组与空白组对比,人脐静脉内皮细胞的EGFL-7表达呈现浓度性抑制作用,有显著差异(P<0.05)。结论：普萘洛尔抑制人脐静脉内皮细胞的增殖,加速细胞早期凋亡,两者呈现浓度依赖性,对成管能力有一定抑制作用,呈现时间依赖性；普萘洛尔成浓度依赖性抑制人内皮细胞中EGFL-7的表达,由此推测普萘洛尔抑制增生期血管瘤作用机制与通过抑制EGFL-7介导的促血管生成作用,最终达到加速血管瘤消退。更多还原

【Abstract】 OUTLINEHemangiomas are the most common congenital benign tumor of infants, occurring in2%-3%of neonates and10%of children<1year of age. Propranolol, a nonselective (3-adrenergic receptor blocker commonly used for arrhythmia, angina pectoris, hypertension. In2008, Leaute-Labreze et al observed that propranolol can control the growth of IHs effectively, especially the proliferation of severe hemangiomas. After that, many groups worldwide have initiated propranolol therapy IHs and it was confirmed that propranolol has significant effective for IHs.A rapid halt in lesion progression and evidence of regression were confirmed in the majority of the cases. Propranolol has become the first-line treatment drug for IHs replaced the hormone. However, there are still a lot of potential side effects, especially in the early of oral propranolol,and required hospitalization to detect heart rate, blood pressure, blood sugar and so on. It has many inconvenience for parents and physicians. In view of this, in chapter Ⅰ-part I:A retrospective chart review was performed on25children (21female and4male) with a median age of4months (range,1-10months). A total of28lesions were treated with1%propranolol ointment. It was showed that Topical therapy with1%propranolol ointment may be a safe and effective method for the treatment of superficial IHs and can be used as an adjuvant treatment measure during the wait-and-see period. In addition, it is currently in dispute that the dosage of propranolol used and the duration of treatment. In chapter Ⅰ-part Ⅱ:A retrospective review (106cases)was performed of IH treated with propranolol at the department of aesthetic, plastic, and burn surgery of the provincial hospital affiliated with Shandong University, Jinan, China, from Sept.16,2009to June1,2013. to describe the dosage of propranolol used, the duration of treatment, the effects and side effects of propranolol treatment. Propranolol administered orally at1to1.5mg/kg/day had a rapid therapeutic effect with few complications.It was unknown that the mechanism of abnormal proliferation on hemangioma. Folkman believed that it was a angiogenesis disease, and the major factor was an uncontrolled angiogenesis in proliferation of hemangioma. The process includes endothelial cell proliferation, extracellular matrix degradation selectivity, increased vascular permeability and migration of cells. The cell growth factor is a class of substances which induce or promote the formation of new blood vessels, regulate endothelial cell proliferation, and have a key role in the ndevelopment of hemangiomas. EGFL7, located in human9q human chromosome, was a chemoattractant for endothelial cellsand is up-regulated in angiogenesis. In chapter Ⅱ-part Ⅱ:The expression of EGFL-7was detected by immuno-histochemical technique in cases of proliferating hemangiomas, involuting hemangiomas, involuted hemangiomas, and normal skins. To detect the expression of EGF-like domain-containing protein-7(EGFL-7) in the different stages of infantile hemangioma, approach the effect and significance of EGFL-7in the occurrence and development of infantile hemangioma. In chapter Ⅱ-part II:Under propranolol intervention, it was observed human umbilical vein endothelial cells (HUVEC) proliferation, apoptosis, lumen formation and expression, EGFL7, to explore the mechanism of inhibition of propranolol hemangioma hyperplasia.Chaper Ⅰ. The efficacy of propranolol treatment of infantile hemangiomaPart Ⅰ. Topical propranolol for treatment of superficial infantile hemangiomasOBJECTIVE:The aim of this study was to evaluate the efficacy and safety of1%propranolol ointment in thetreatment of superficial IHs.METHODS:A retrospective chart review was performed on25children (21female and4male) with a median age of4months (range,1-10months). A total of28superficial IHs were treated with1%propranolol ointment. Topical propranolol was applied thrice daily for a mean duration of21weeks (range,5-59weeks). Changes in the size, texture, and color of the tumor were monitored and recorded at regular intervals. The treatment response was evaluated using a3-point scale system: good, partial, and no response. Adverse effects after medication were evaluated and managed accordingly.RESULTS:Of the28hemangiomas,16(57%) demonstrated good response,17(34.7%) showed a partial response, and3(10%) had no response. Among all the IHs,90%(95%confidence interval72%-98%) showed either good or partial responses to topical1%propranolol ointment treatment. No systemic complication was observed in any of the patients.CONCLUSION:Topical therapy with1%propranolol ointment may be a safe and effective method for the treatment of superficial IHs and can be used as an adjuvant treatment measure during the wait-and-see period. Part Ⅱ Oral propranolol in the management of infantile hemangiomaOBJECTIVE:to describe the side effects of propranolol treatment in infantile hemangiomas(IHs).METHODS:A retrospective chart review was conducted on all children who attended clinic from Sept.16,2009to Nov.11,2013.106children (71girels and35boys, mean age and weight at onset of treatment:5.1months and7.3kg). Prorpanolol was administered in a progressive schedule to1.0to1.5mg/kg per day, divided in three doses. Demographic data, clinical features, imaging findings treatment regimens of propranolol, and outcome were investigated. Adverse effects after medication were evaluated and managed accordingly.RESULTS:All106patients had a good response. Side effects that needed intervention and/or close monitoring were included diarrhea (n=10), hypotension (n=7), and milk regurgitation (n=2). Nightmares (n=2), agitation (n=1), and cold extremities (n=1) were observed.CONCLUSIONS:Propranolol administered orally at1tol.5mg/kg/day had a rapid therapeutic effect with few complications. Chaper Ⅱ. The experimental study of propranolol treatment of infantile hemangiomaPart Ⅰ. The expression and significance of EGFL-7in infantile hemangiomaOBJECTIVE:To detect the expression of EGF-like domain-containing protein-7(EGFL-7) in the different stages of infantile hemangioma, approach the effect and significance of EGFL-7in the occurrence and development of infantile hemangioma.METHODS:Collect infantile hemangioma specimens embedded in paraffin, the specimens were all excised by operations and were confirmed by pathologic diagnosis. After routinely HE stain, we classified the specimens according to the Mulliken’s classification standard, including proliferating group, involuting group and involuted group, normal skin tissues were chosen as control group. The expression of EGFL-7was detected by immuno-histochemical technique in cases of proliferating hemangiomas, involuting hemangiomas, involuted hemangiomas, and normal skins; the mean optical density was measured by image analysis system.RESULTS:Positive expression of EGFL-7was located in cytoplasm tumor cells and nomal skin tissues. The strongly positive or positive expression in proliferating phase, positive expression in involuting phase, and weakly positive or negative expression in involuted phase and normal skin tissuesCONCLUSION:EGFL-7are involved in the occurrence and development of infantile hemangioma and there is a close relationship between them. Part Ⅱ. The effect of propranolol in biological behavior of and expression of EGFL-7of HUVECOBJECTIVE:To investigate effects of propranolol in biological behavior and EGFL-7of the human umbilical vein endothelial cells in vitro. It would be provided a theoretical basis for further study of propranolol for infantile hemangiomas mechanism.METHODS:The human unbilical vein endothelial cell line was cultered by propranolol with varying concentration. Cell growth was assessed by MTT, the apoptotic rate was assessed by Annexin V-FITC/PI double staining method, the tube formation ability was assessed by tube formation assay. The expression of EGFL-7mRNA and protein were identified by semi-quantitive reverse transcriptase polymerase chain reaction(RT-PCR) and western blot, respectively.RESULTS:After drug processing for24h and48h, compared with the normal control group, the inhibition rates of25μmol/L,50μmol/L,100μmol/L,125μmol/L propranolol on proliferation of HUVEC were found an upward trend (P<0.01) and then we could acquire suitable concentration by calculated IC50=125.33±2.71μm. After cultered for24h, the early apoptotic rates of Oμmol/L,25μmol/L,125μmol/L were detected2.7665±0.1528(%),15.467±2.3861(%),26.133±1.6503(%)(P<0.05). The tube formation capacity were not significant differences after drug processing (Oμmol/L,25μmol/L,125μmol/L) for24hours, and after48h, it was found that the differences of tube formation capacity. Propranolol can down-regulate the expression of EGFL-7mRNA and protein in a concentration-dependent manner(P<0.05).CONCLUSION:Propranolol inhibited HUVEC proliferation and promoted the apoptosis in a concentration dependent manner, and inhibited the tube formation of HUVEC in a time-dependent manner. EGFL-7are involved in the occurrence and development of infantile hemangioma and there is a close relationship between them.更多还原