【作者】 魏艇；

【导师】 刘文英；

【作者基本信息】 四川大学 ， 外科学， 2007， 博士

【摘要】 第一部分人血管瘤裸小鼠移植模型的建立和鉴定背景和目的：婴幼儿血管瘤是小儿最常见的良性肿瘤。大多数血管瘤可以自行消退而不需要进行任何干预。但是大约有10％的血管瘤由于其生长过于迅速或者生长部位特殊而可能导致毁容、溃疡、畸形、功能障碍甚至危及生命，故需要积极的医疗干预。目前血管瘤的治疗方法很多，但是没有任何一种治疗方法对所有血管瘤有效，而且治疗本身也可能发生一些严重的并发症。多年来寻找到有效、安全治疗血管瘤的方法一直是科研工作者和临床医生们孜孜不倦奋斗的目标。而之所以有目前治疗的窘境就是因为对血管瘤增生和消退的机制还认识不清。缺乏理想的研究模型是阻碍对血管瘤病理机制深入探讨的主要原因之一。本科研小组利用人体血管瘤组织块裸小鼠皮下移植建立人体血管瘤裸小鼠移植模型，来初步探讨婴幼儿血管瘤发生发展的可能机制。方法：人体血管瘤取自1例2月龄男性患儿左小腿外侧皮肤和皮下血管瘤。将手术切除的人体血管瘤标本的皮下部分，切成5mm×4mm×3mm的小块，分别经裸小鼠的皮肤切口将血管瘤组织块置于项部、腰背部和双侧腋下的皮下，每只4处。于移植后第1、7、14、21、28、35、42、49、56、63、70、90、120、180天用游标卡尺测量肿瘤最大横径a和纵径b，定期观察移植瘤体的生长状况。分别于移植后56、120、180天各取2个移植成活瘤体作HE、CD31、Glut1染色。使用Nikon E600研究显微镜对切片进行观察，用SPOT Cool CCD摄像头进行图像采集。用Image pro plus 4.5版本的专业图像分析软件进行图像分析。测定在单位视野中免疫组织化学反应阳性颗粒的面积、平均光密度以及积分光密度。采用SPSS11.0统计软件进行统计学分析，结果用（?）±SD表示，对免疫反应阳性颗粒积分光密度进行t检验，检验水准为0.05。结果：血管瘤组织块移植后3～4周内，瘤体基本上无明显变化；42天后，大多数瘤体开始生长，体积较移植时增大，瘤体周围可见新生毛细血管；56天左右瘤体迅速增大，生长良好，透过皮肤见瘤体发蓝，较有弹性；63天左右达到最大；之后瘤体体积保持不变或轻微缩小，至90天时可见部分瘤体颜色变浅，体积缩小，质地较前变硬，180天时少数瘤体残留，呈淡黄色，质地变硬，剖面呈纤维脂肪组织。HE染色发现增生期移植血管瘤内大量血管瘤内皮细胞增殖堆积，聚集成团巢状，血管腔隙小而不规则；消退期移植血管瘤内血管瘤内皮细胞明显减少，血管腔扩大，内衬扁平的内皮细胞，大量纤维脂肪组织沉积。免疫组织化学检测显示：移植成活的血管瘤CD31呈阳性表达，CD31定位于血管内皮细胞的细胞膜上。移植成活的血管瘤Glut1呈强阳性表达，Glut1定位于血管瘤内皮细胞的细胞膜上。移植供体人血管瘤的血管内皮细胞Glut1染色也呈强阳性。结论：本研究将人体血管瘤组织块移植到裸小鼠皮下，成功建立了人体血管瘤裸小鼠移植模型，并且通过观察移植瘤的生长状况、HE染色和免疫组织化学检测从多方面证实了该模型能客观真实再现人体血管瘤增生和消退的过程，为后续试验奠定了良好的动物模型基础。第二部分血管生成素-1、血管生成素-2及Tie2受体在人体血管瘤裸小鼠移植模型中的表达背景和目的：婴幼儿血管瘤是以血管内皮细胞的异常增殖和血管异常生成为特点。Mulliken根据血管瘤组织学特点将其分为增生期、消退期和消退完成期。血管瘤如何从迅速增生扩大转变为自发缓慢消退，其潜在的分子与生物化学机制目前仍认识不清。血管瘤与一般良性肿瘤有着明显不同之处就是大多数的婴幼儿血管瘤可以自行消退，故血管瘤是否为真性肿瘤引起了人们的争议。近年来，有众多研究显示血管生成素及其Tie2受体家族在正常以及异常血管生成中扮演重要角色，故我们利用已建立的人体血管瘤裸小鼠移植模型来研究血管生成素-1、血管生成素-2以及Tie2受体在人体血管瘤增生与消退时期的动态表达情况。方法：动物模型建立见第一部分。分别于移植后56天、120天各切取12个移植存活瘤体。其中2个瘤体用4％多聚甲醛固定，作免疫组织化学检测（Ang1，Ang2，Tie2和VEGF）；5个瘤体用Trizol液固定后低温转运作RT-PCR荧光定量检测（Angl，Ang2和Tie2）；5个瘤体低温转运作Western blot检测（Ang1，Ang2和Tie2）。另取正常皮肤组织（包茎患儿包皮环切术切除的包皮组织）5块用于荧光定量RT-PCR，Western blot和免疫组织化学检测。使用REST软件将荧光定量RT-PCR所得Ct值转换为2^-△△Ct值后进行统计学分析。免疫组织化学检测结果的图像采集和统计学分析同第一部分。结果：荧光定量RT-PCR和Western blot的结果显示在增生期和消退期移植瘤中，Ang1mRNA和蛋白的表达量明显低于正常包皮组织；Ang2和Tie2mRNA和蛋白的的表达量明显高于正常包皮组织。三者的表达在增生期和消退期移植瘤之间没有统计学差异。免疫组织化学检测结果显示Ang2和Tie2在增生期和消退期移植瘤中表达呈强阳性，分别定位于血管内皮细胞的细胞浆和细胞膜上，在正常包皮组织中Ang2和Tie2的表达呈弱阳性。在增生期和消退期移植瘤中，Ang1表达呈弱阳性，定位于血管内皮细胞的细胞膜和血管周围细胞的细胞浆内，在正常包皮组织中Ang1的表达呈阳性。在VEGF免疫组化染色的切片上，发现在血管瘤组织内存在血管发生（vasculogenesis）这种原始的血管生成方式，这种血管生成方式只有在胚胎时期和病理状况下可以出现。结论：婴幼儿血管瘤中存在血管生成素-1、血管生成素-2和Tie2受体的异常表达，提示婴幼儿血管瘤可能并非一种真性血管肿瘤，而是一种血管生成异常的疾病。更多还原

【Abstract】 PartⅠA New Human Hemangioma Xenograft Model on Nude MiceBackground and Objective: Infantile hemangioma(often abbreviated as hemangioma) is the most common benign tumor involving infants characterized by appearance during the first weeks of life, rapid growth for 6～10 months followed by spontaneous and slow regression in the subsequent 1～5 years. Though most of infantile hemangiomas can regress spontaneously, about 10％hemangiomas can bring disasters to infants because of rapid growth or special location, such as ulceration, hemorrhage, disfiguration and even life threatening. Up to date, there are all kinds of therapeutic strategies including corticosteroid, interferon-α, surgery, laser, and so on. But none of them is perfect because of limited effectiveness and side effects. The reason of clinical dilemmas lies in lack of adequate knowledge of pathogenesis of hemangioma. To investigate the underlying mechanisms of hemangioma requires a hemangioma model that can present the natural development and characteristics of human hemangioma without deviation from ethics. Unfortunately, currently available experimental models cannot meet the requirements satisfactorily and hinder researchers’ footsteps toward. In this research we attempted to transplant human hemangioma into nude mice subcutaneously to establish a xenograft animal model of human hemangioma for the future investigation.Methods: An infantile hemangioma was obtained from a male baby aged 2 months according to a protocol approved by Westchina Hospital Ethics Committee. The pathological diagnosis was confirmed with Mulliken’s classification.8 BALB/c nu/nu nude mice were used in this experiment. The hemangioma specimen was cut into small blocks of 5mm×4mm×3mm in size. The blocks were transplanted into 8 nude mice subcutaneously, 4 blocks each mouse. The volume of the grafted hemangiomas was measured with a vernier on day 1, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 90, 120 and 180 after transplantation. The growth of the grafts was observed on regular basis. 2 grafted hemangiomas were harvested on day 56, 120 and 180 after transplantation respectively. Formaldehyde-fixed and paraffin-embedded specimens were sliced into 5μm sections for HE staining and immunohistochemistry for CD31(monoclonal mouse anti-human antibody, Neomarkers, USA) and Glut1(polyclonal rabbit anti-human antibody, Neomarkers, USA).The slices were observed under Nikon E600 microscope. The pictures were taken with a SPOT Cool CCD camera and analyzed with Image pro plus 4.5 software. IOD(integrated optical density) was chosen to represent staining intensity and presented as (?)+SD. SPSS11.0 software was used to perform t text.Results: The volume changes of the grafts were not obvious in the first 3-4 weeks. Later on some of the grafts became bigger and bigger and reached the apex at the end of 2nd month after transplantation with new vessel sprouts on the periphery. After 90 days the grafts began to shrink and became pale and yellow with the texture hardening. On day 180 only a few grafts remained and fibrofatty tissue was found at dissection.HE staining showed the proliferating graft hemangiomas were crowded with a large number of plump vascular endothelial cells(VEC) with small and irregular vessel lumens. In the involuting graft hemanigoms, the VECs were sharply decreased with enlarged vessel lumens lined with flat VECs. A great deal of fibrofatty tissue was deposited in the grafts.Immunohistochemisty showed the graft hemangiomas were positive with CD31 and Glut1 staining. Both CD31 and Glut1 were located on cell membrane.Conclusion: A human hemangioma xenograft aninal model on nude mice is successfully established by transplanting human hemangioma tissue blocks into nude mice subcutaneously. And we testifies the validity of the animal model by morphologic observation, HE staining and immunohistochemistry. This model provides an excellent basis for future research. PartⅡThe Expression of Angiopoietin-1, Angiopoietin-2 and Tie2 in Human Hemangioma Xenograft Animal ModelBackground and Objective: Infantile hemangioma is characterized by abnormal vascular endothelial cell proliferation and disturbed angiogenesis. Mulliken categorized infantile hemangioma into three subgroups: proliferating, involuting and involuted hemangioma. The underlying molecular and biochemical mechanisms by which proliferating hemangiomas begin to regress spontaneously and slowly is unclear up to date. A distinctive characteristic of infantile hemangioma from other tumors is that 80％of hemangiomas can regress spontaneously which has caused a widespread dispute that is whether infantile hemangioma is a real tumor. In the past decade, many literatures have shown that the angiopoietin/Tie2 pathway plays an important role in normal and abnormal angiogenesis. In this research, we attempted to detect the dynamic expression of angiopoietin-1, angiopoietin-2 and Tie2 in different phases of hemangioma by means of the previously established animal model. Mothods: The animal models were established according to PartⅠ. Twelve specimens were harvested on day 56 and 120 after grafting respectively. 2 of 12 were fixed with 4％formaldehyde for immunohistochemistry for Ang1(goat anti-human antibody, RnD, USA), Ang2(goat anti-human antibody, RnD, USA), Tie2(goat anti-human antibody, RnD, USA) and VEGF(rabbit anti-human antibody, Boster, China). 5 of 12 were fixed with Trizol for fluorescence RT-PCR for Angl, Ang2 and Tie2. The remains were for Western blot analysis for Angl, Ang2 and Tie2. 5 normal foreskins were acquired from 5 boys(undergoing circumcision) for immunohistochemistry, RT-PCR and Western blot.Statistical analysis was performed with REST software for the RT-PCR results and SPSS11.0 software for immunohistochemistry results.Results: Fluorescence RT-PCR and Western blot showed the amount of Ang1mRNA and protein in the proliferating and involuting graft hemangiomas was much less than that of the normal foreskins. On the contrary, the amount of mRNA and protein of Ang2 and Tie2 in the proliferating and involuting graft hemangiomas was much more than that of the normal foreskins. There was no statistical difference between proliferating and involuting phases in Ang1, Ang2 and Tie2. Immunohistochemistry showed a similar result with RT-PCR and Western blot. Vasculogenesis was found in the graft hemangiomas which was a primary process of vessel formation only occurring during embryogenesis and under a pathologic condition.Conclusion: Abnormal expression of Ang1, Ang2 and Tie2 is found in infantile hemangiomas. A hypothesis is forwarded that it is possible that infantile hemangioma is not a real tumor, but rather a disorder of vasculogenesis and angiogenesis.更多还原