利用RNA干扰技术下调血管瘤中VEGF基因的表达对血管瘤影响的实验研究
|
摘要
血管瘤是婴幼儿最常见的皮肤肿瘤,好发于头面部和四肢等体表部位,是以血管内皮细胞增生为主的良性肿瘤。其特点是在婴幼儿出生后第1年迅速增长,在随后的7-9年缓慢消退。一般将其分为增殖期、消退期和消退后期3个阶段。其中相当一部分血管瘤可以自然消退,不留下任何后遗症;还有部分血管瘤可能破溃感染留下瘢痕,大约20%血管瘤会破坏正常组织甚至危及生命。但是其发病机理和消退机制到目前还不清楚。因此血管瘤的治疗方法有很多,但是疗效却不确切。研究其发病机制不仅能够预防血管瘤的发生,同时也有助于临床的治疗。
目前研究发现,有助于血管生成的因子有:血管内皮细胞生长因子(VEGF)、雌二醇(oestradiol)、成纤维生长因子(FGF)、血管生长素(angiogenin)、转化生长因子(TGF)、α-肿瘤坏死因子(TNF-α)、血小板衍生生长因子(PDGF)、白介素-8(IL-8)等。其中VEGF被认为与血管瘤的生成、发展和消退有着重要的联系。VEGF具有促进血管化、维持内皮细胞的活性、促进内皮细胞的迁移、促进内皮细胞的增殖、增加微血管通透性等特性。它是内皮细胞(Endothelial cell,ECs)的特异性有丝分裂原,能选择性的作用于EC,促进其增殖、迁移,有利于血管生成。在肿瘤、缺血缺氧等情况下,VEGF及其受体呈高表达。而VEGF不足时会导致血管管腔闭合、血管退化。
目前利用基因干扰技术下调VEGF的表达在研究与治疗肿瘤方面取得了极大的进展。同时有学者发现增殖性血管瘤(婴幼儿血管瘤,IH)不同于其它类型的血管畸形,其早期迅速的扩张是血管内皮细胞的非控性增生,这些增生是血管形成刺激因子或抑制因子水平异常引起的。其中VEGF被认为在这种血管瘤的形成中起十分重要的作用。将VEGF作为一种新的治疗靶点,已部分应用到血管瘤的临床诊断和治疗中。
本课题拟利用基因干扰技术,通过体内和体外实验研究,下调血管瘤动物模型和婴幼儿血管瘤血管内皮细胞中VEGF基因的表达,观察血管瘤和血管瘤内皮细胞的发展变化,同时探讨血管瘤的增殖和消退机制。为进一步临床应用研究奠定理论基础。
1.裸鼠血管瘤移植模型的建立
目的:建立人毛细血管瘤裸鼠移植模型,探讨血管瘤裸鼠模型建立的最佳条件。方法:将手术切除的雌激素受体阳性的儿童增生期血管瘤组织制成组织块,植入20只裸鼠(BALB/c nude mice)皮下,每只4处,将20只裸鼠分为4个实验组。实验1组在移植后给予普通鼠食喂养;实验2组在1组基础上每周肌注雌二醇0.01 mg;实验3组在1组基础上每周肌注雌二醇0.1 mg;实验4组在1组基础上每周肌注雌二醇1mg,于移植后第30、60、90天切取移植瘤。移植瘤标本进行病理学光镜检查,用血管内皮细胞单克隆抗体CD31、CD34、Ki-67行免疫组化染色。结果:移植后早期各组标本内皮细胞大量变性、坏死,30d后,单纯喂养的实验1组及实验2组部分移植瘤开始吸收或形成脓肿及纤维化。实验3、4组移植瘤开始缓慢生长。90d后实验1组实验2组移植瘤均未成活,实验4组移植瘤部分成活,而实验3组移植瘤全部成活。光镜下成活的移植瘤与原血管瘤组织生物学特点相似。结论:不同剂量的雌激素对血管瘤裸鼠移植模型的建立有一定影响,适量的应用雌激素可建立稳定的人血管瘤裸鼠移植动物模型。该模型可以应用到基础和临床的血管瘤研究。
2.增殖期血管瘤内皮细胞的培养和鉴定
目的:体外培养和鉴定血管瘤内皮细胞(Hemangioma endothelial cell,HemEC)。方法:无菌条件下手术切取增生期血管瘤组织标本,利用组织块法进行培养。用含20%胎牛血清的M199培养基,加入内皮细胞生长支持物(Endothelial cell growth supplement,ECGS,浓度为100μg/ml)在含5%CO2、37℃培养箱中培养,每隔两到三天换液,细胞铺满瓶底后传代。用光学显微镜观察细胞形态和生长情况,并用免疫组织化学检测细胞表达Ⅷ因子相关抗原和透射电镜检测鉴定细胞。结果:组织块接种3天后有细胞开始从边缘移出,细胞成多角形,1周左右混合成片,部分汇合成岛状。大约3周可以铺满瓶底,随后消化传代。传代细胞成典型的“鹅卵石”样排列铺满瓶底。细胞Ⅷ因子染色为阳性,透射电镜显示细胞质内含有ECs特征性的Weibel-Palade小体(W-P小体,呈点状或卵形)。结论:利用组织块法可以获得纯度较高的血管瘤内皮细胞,该方法简单易用。
3.VEGF基因干扰质粒对血管瘤内皮细胞的影响
目的:应用人血管内皮生长因子(VEGF)干扰质粒,作用于体外培养的人增生期血管瘤内皮细胞,观察其对VEGF分泌及对血管瘤内皮细胞的影响。方法:利用脂质体法将真核表达质粒pGCsi-U6NeoRFP-shVEGF转染到HemECs。采用倒置荧光显微镜观察质粒转染效果;用MTT法和酶联免疫吸附(ELISA)法分析观察shVEGF对HemECs的影响。结果:转染组的HemECs与对照组相比, VEGF表达明显减少,转染组培养上清中的VEGF表达量在转染的第1、3、5、7天分别为141.3±7.82、92.34±5.71、68.07±5.95及40.65±6.71pg/ml于其它组比较P<0.05,且VEGF质粒的转染使VEGF的表达下降也促进了细胞的凋亡。结论:转染VEGF干扰质粒可以明显减少血管瘤内皮细胞VEGF的表达,同时也促进了细胞的凋亡。
4.VEGF干扰慢病毒对裸鼠血管瘤移植模型的影响
目的:制作VEGF慢病毒干扰载体Lenti-siRNA-VEGF,将其作用于裸鼠血管瘤移植模型,观察血管瘤的生长和凋亡的变化,探讨新的血管瘤防治方法。方法:将VEGF干扰质粒包装成慢病毒载体的Lenti-siRNA-VEGF。制作裸鼠血管瘤移植模型,将Lenti-siRNA-VEGF作用于移植模型,大体观察瘤体变化,利用HE、免疫组化和TUNEL等方法观察瘤体切片。再用Western-blot方法从蛋白质水平探讨VEGF被沉默后血管瘤的生长和消退情况。结果:实验组瘤体在注射后1周瘤体体积开始变小,1个月后瘤体缩小变硬;对照组无明显变化。HE检测可见管腔闭塞,瘤体纤维化。CD31、CD34免疫组化检测发现阳性细胞数分别为7.68±0.83、10.25±1.29(P<0.05)。TUNEL检测发现细胞凋亡较对照组明显增多。Western-blot发现实验组VEGF蛋白表达明显下降。结论:Lenti-siRNA-VEGF作用于移植模型可以明显降低瘤体VEGF的表达,促进瘤体内皮细胞凋亡。该方法为血管瘤进一步实验和临床研究提供一个新的方法。
Hemangioma is one of the most prevalent skin neoplasia which is usually occur in facial , head and limb skin for infants and children ,and it is a benign tumor take the vascular endothelial cells proliferation as the main characteristic. The hemangioma grows very fast after been born 1 year later ,and will disappear in the following 7-9 years. And generally it is been divided into three phases, the proliferation, the subside, and the post subside phase, most of the hemangioma can fade away naturally without any trace, but some of the hemangioma may rupture and cause inflammation and then there will be scar left, and there still 20% of hemangioma will destroy normal tissue even endanger human life. The mechanism how it occur and disappear is still unknown and now there is many therapies to do the treatment but the effect is not clear. To research the mechanism not only can prevent hemangioma incidence but also do good for clinic treatment .
The latest research found that there are many factor can promote angiogenesis including VEGF, oestradiol, FEG, angiogenin, TGF, TNF-α, PDGF, IL-8 etc .And VEGF was considered as a very important factor involving angiogenesis, developing and fading away. The VEGF can promote angiogenesis, keeping the activation of endothelial cells, helping migration of endothelial cells, promote its proliferation, enhance the permeability of microvessel and so on. The VEGF is an very critical mitogen for endothelial cells, which can specifically work on endothelial cells to promote proliferation, migration to enhance angiogenesis. Under the condition of tumor and hypoxia or ischemia, VEGF expression and its receptors are unregulated. with the VEGF deletion, there will be lumen closure and degeneration .
By taking use of gene interference to down regulating the expression of VEGF to research and do the therapy has gain significant progress, at the same time the researchers has found that proliferating hemangioma (IH) is different from other types of blood malformation, and the earlier stage growth is caused by uncontrollable ECs, and VEGF was considered as a key factor in the process, taking VEGF as a new target for therapy to cure hemagioma is partly using in diagnosis and clinical treatment .
This research work using RNA interference technique to down regulate VEGF expression level in hemangioma mouse model and infant hemangioma ECs in vivo and in vitro respectively to investigate hemangioma and hemangioma ECs activity and development, also to detect the mechanism of hemangioma proliferation and fading away, in order to provide solid evidence for future clinical therapy.
1. The modeling of nude mice hemangioma transplantation
Aim: To establish the nude mice to mentum hemangioma transplantation model to evaluate the best condition. Method: Taking the surgery cutting estrogen receptor positive hemangioma tissue at the stage of proliferation from children to plant the tissues under the skin of 20 BALB/c nude mice, four sites on every nude mouse, and then divided these mice into four groups, group1 was fed with normal food after transplantation, group 2 was injected with estrogen 0.01 mg every week at the basis of group1, group3 was injected with estrogen 0.1mg every week at the basis of group 1, and group 4 was injected with estrogen 1mg every week, after 30,60,90 days respectively, to harvest the transplantation hemangioma and then them into pathological examination by microscope, and also did the immunohistochemistry staining by CD31, CD34,Ki-67 monoantibody to these samples. Results: At the earlier stage after transplantation there were large sums of ECs degeneration and necrosis, part of the transplantation hemangioma began to absorb and being state of fibrosis in the group1 and group2 after 30 days, and in the group3 and group4 the transplantation tissue began to proliferate gradually. After 90 days we found that there were no ECs survival in the group1 and group2, part of hemangioma survived in the group4, and in the group3, all the transplantation hemangioma survived. Under the microscope, there are similarities compare with the original hemangioma. Conclusion: Different does of estrogen has different effect on the establish of the nude mice hemangioma transplantation model, proper does estrogen can help to establish stable human hemangioma nude mice transpaltation model, which could be taken into preclinical and clinical research .
2. The culture and identification of proliferating hemangioma ECs in vitro
Aim: To culture and identify the hemangioma endothelial cell(HemEC). Methods: To collect the surgery cutting proliferating hemangioma tissue under the condition of sterile, and using the tissue to culture. By using the M199 medium with 20% FBS adding endothelial cell growth supplement (ECGS 100μg/ml) in the 5%CO2、37℃incubator, we changed the medium every 2 -3 days, and passage them until the bottle of the cell culture bottle is full. To watch the growth and morphological changes by the microscope and to identify the cells by the TEM and to test theⅧfactor associated antigen by immunohistochemistry. Results: There were cell migration from the edge of the tissue after 3 days of plantation, cells became polygon, after 1 week the cells mixed into pieces and part of the cells mix like isles .About 3 weeks later the bottle bottom was full and then passage the cells, there were typical pebble on the bottom, theⅧfactor associated antigen was positive by immunohistochemistry the TEM showed there were Weibel-Palade bodied which was the characteristic of ECs in the cells. Conclusion: By using the explants could acquire relatively pure hemangioma Ecs, and this method was easy and effective.
3. The effect of VEGF interference plasmid on hemangioma ECs
Aim: By using VEGF interference plasmid to work on the proliferating hemangioma ECs and watch the VEGF secretion and its effect. Method: Transfection of expression plasmid pGCsi-U6NeoRFP-shVEGF into HemECs, to watch the transfection efficiency by the Inverted fluorescence microscope; to exam the effect of sh VEGF to HemECs by MTT and ELISA. Results: Compared with the control group, the transfected HemECs VEGF expression lever decreased significantly, in the medium of the transfected group, the expression of VEGF in the 1st, 3rd, 5th and 7th day is 141.3±7.82、92.34±5.71、68.07±5.95 and 40.65±6.71pg/ml(P < 0.05), respectively, and the down regulation of VEGF also promoted cell apoptosis. Conclusion: Transfection of the VEGF interference plasmid could decrease the VEGF expression significantly in the HemECs and also promote apoptosis.
4. The effect of VEGF interference lentivirus on the nude mice hemangioma transplantation model
Aim: To establish the VEGF interference lentivirus Lenti-siRNA-VEGF, then work it on the nude mice hemangioma transplantation model to watch the growth and apoptosis of the hemangioma in order to discover new therapy for it. Methods: We constructed Lenti-siRNA-VEGF and established. The nude mice hemangioma transplantation model and then took Lenti-siRNA-VEGF in use on the transplantation model to watch the morphological changes, taking HE, immunohistochemistry and TUNEL to watch the hemangioma sections. By using western blotting to evaluate the growth of hemagioma and fading away statement after VEGF was down regulated. Results: The hemangioma began to became smaller after injection of Lenti-siRNA-VEGF 1 week later, and one month later the body of the hemangioma began to reduce and hardening, at the same time the control group without changes. HE staining showed that vessel space closed up and hemangioma fibrosis. CD31, CD34 immunohistochemistry showed that the positive cells were 7.68±0.83, 10.25±1.29(P<0.05) respectively. TUNEL assay found that there were more apoptosis cell in the experiment group. Western blotting showed that VEGF protein expression decreased significantly. Conclusion: The Lenti-siRNA-VEGF worked on transplantation model can down regulating the VEGF significantly, also promoted HemECs apoptosis. This method supplied an effective way for further experiment and clinical research.
目前研究发现,有助于血管生成的因子有:血管内皮细胞生长因子(VEGF)、雌二醇(oestradiol)、成纤维生长因子(FGF)、血管生长素(angiogenin)、转化生长因子(TGF)、α-肿瘤坏死因子(TNF-α)、血小板衍生生长因子(PDGF)、白介素-8(IL-8)等。其中VEGF被认为与血管瘤的生成、发展和消退有着重要的联系。VEGF具有促进血管化、维持内皮细胞的活性、促进内皮细胞的迁移、促进内皮细胞的增殖、增加微血管通透性等特性。它是内皮细胞(Endothelial cell,ECs)的特异性有丝分裂原,能选择性的作用于EC,促进其增殖、迁移,有利于血管生成。在肿瘤、缺血缺氧等情况下,VEGF及其受体呈高表达。而VEGF不足时会导致血管管腔闭合、血管退化。
目前利用基因干扰技术下调VEGF的表达在研究与治疗肿瘤方面取得了极大的进展。同时有学者发现增殖性血管瘤(婴幼儿血管瘤,IH)不同于其它类型的血管畸形,其早期迅速的扩张是血管内皮细胞的非控性增生,这些增生是血管形成刺激因子或抑制因子水平异常引起的。其中VEGF被认为在这种血管瘤的形成中起十分重要的作用。将VEGF作为一种新的治疗靶点,已部分应用到血管瘤的临床诊断和治疗中。
本课题拟利用基因干扰技术,通过体内和体外实验研究,下调血管瘤动物模型和婴幼儿血管瘤血管内皮细胞中VEGF基因的表达,观察血管瘤和血管瘤内皮细胞的发展变化,同时探讨血管瘤的增殖和消退机制。为进一步临床应用研究奠定理论基础。
1.裸鼠血管瘤移植模型的建立
目的:建立人毛细血管瘤裸鼠移植模型,探讨血管瘤裸鼠模型建立的最佳条件。方法:将手术切除的雌激素受体阳性的儿童增生期血管瘤组织制成组织块,植入20只裸鼠(BALB/c nude mice)皮下,每只4处,将20只裸鼠分为4个实验组。实验1组在移植后给予普通鼠食喂养;实验2组在1组基础上每周肌注雌二醇0.01 mg;实验3组在1组基础上每周肌注雌二醇0.1 mg;实验4组在1组基础上每周肌注雌二醇1mg,于移植后第30、60、90天切取移植瘤。移植瘤标本进行病理学光镜检查,用血管内皮细胞单克隆抗体CD31、CD34、Ki-67行免疫组化染色。结果:移植后早期各组标本内皮细胞大量变性、坏死,30d后,单纯喂养的实验1组及实验2组部分移植瘤开始吸收或形成脓肿及纤维化。实验3、4组移植瘤开始缓慢生长。90d后实验1组实验2组移植瘤均未成活,实验4组移植瘤部分成活,而实验3组移植瘤全部成活。光镜下成活的移植瘤与原血管瘤组织生物学特点相似。结论:不同剂量的雌激素对血管瘤裸鼠移植模型的建立有一定影响,适量的应用雌激素可建立稳定的人血管瘤裸鼠移植动物模型。该模型可以应用到基础和临床的血管瘤研究。
2.增殖期血管瘤内皮细胞的培养和鉴定
目的:体外培养和鉴定血管瘤内皮细胞(Hemangioma endothelial cell,HemEC)。方法:无菌条件下手术切取增生期血管瘤组织标本,利用组织块法进行培养。用含20%胎牛血清的M199培养基,加入内皮细胞生长支持物(Endothelial cell growth supplement,ECGS,浓度为100μg/ml)在含5%CO2、37℃培养箱中培养,每隔两到三天换液,细胞铺满瓶底后传代。用光学显微镜观察细胞形态和生长情况,并用免疫组织化学检测细胞表达Ⅷ因子相关抗原和透射电镜检测鉴定细胞。结果:组织块接种3天后有细胞开始从边缘移出,细胞成多角形,1周左右混合成片,部分汇合成岛状。大约3周可以铺满瓶底,随后消化传代。传代细胞成典型的“鹅卵石”样排列铺满瓶底。细胞Ⅷ因子染色为阳性,透射电镜显示细胞质内含有ECs特征性的Weibel-Palade小体(W-P小体,呈点状或卵形)。结论:利用组织块法可以获得纯度较高的血管瘤内皮细胞,该方法简单易用。
3.VEGF基因干扰质粒对血管瘤内皮细胞的影响
目的:应用人血管内皮生长因子(VEGF)干扰质粒,作用于体外培养的人增生期血管瘤内皮细胞,观察其对VEGF分泌及对血管瘤内皮细胞的影响。方法:利用脂质体法将真核表达质粒pGCsi-U6NeoRFP-shVEGF转染到HemECs。采用倒置荧光显微镜观察质粒转染效果;用MTT法和酶联免疫吸附(ELISA)法分析观察shVEGF对HemECs的影响。结果:转染组的HemECs与对照组相比, VEGF表达明显减少,转染组培养上清中的VEGF表达量在转染的第1、3、5、7天分别为141.3±7.82、92.34±5.71、68.07±5.95及40.65±6.71pg/ml于其它组比较P<0.05,且VEGF质粒的转染使VEGF的表达下降也促进了细胞的凋亡。结论:转染VEGF干扰质粒可以明显减少血管瘤内皮细胞VEGF的表达,同时也促进了细胞的凋亡。
4.VEGF干扰慢病毒对裸鼠血管瘤移植模型的影响
目的:制作VEGF慢病毒干扰载体Lenti-siRNA-VEGF,将其作用于裸鼠血管瘤移植模型,观察血管瘤的生长和凋亡的变化,探讨新的血管瘤防治方法。方法:将VEGF干扰质粒包装成慢病毒载体的Lenti-siRNA-VEGF。制作裸鼠血管瘤移植模型,将Lenti-siRNA-VEGF作用于移植模型,大体观察瘤体变化,利用HE、免疫组化和TUNEL等方法观察瘤体切片。再用Western-blot方法从蛋白质水平探讨VEGF被沉默后血管瘤的生长和消退情况。结果:实验组瘤体在注射后1周瘤体体积开始变小,1个月后瘤体缩小变硬;对照组无明显变化。HE检测可见管腔闭塞,瘤体纤维化。CD31、CD34免疫组化检测发现阳性细胞数分别为7.68±0.83、10.25±1.29(P<0.05)。TUNEL检测发现细胞凋亡较对照组明显增多。Western-blot发现实验组VEGF蛋白表达明显下降。结论:Lenti-siRNA-VEGF作用于移植模型可以明显降低瘤体VEGF的表达,促进瘤体内皮细胞凋亡。该方法为血管瘤进一步实验和临床研究提供一个新的方法。
Hemangioma is one of the most prevalent skin neoplasia which is usually occur in facial , head and limb skin for infants and children ,and it is a benign tumor take the vascular endothelial cells proliferation as the main characteristic. The hemangioma grows very fast after been born 1 year later ,and will disappear in the following 7-9 years. And generally it is been divided into three phases, the proliferation, the subside, and the post subside phase, most of the hemangioma can fade away naturally without any trace, but some of the hemangioma may rupture and cause inflammation and then there will be scar left, and there still 20% of hemangioma will destroy normal tissue even endanger human life. The mechanism how it occur and disappear is still unknown and now there is many therapies to do the treatment but the effect is not clear. To research the mechanism not only can prevent hemangioma incidence but also do good for clinic treatment .
The latest research found that there are many factor can promote angiogenesis including VEGF, oestradiol, FEG, angiogenin, TGF, TNF-α, PDGF, IL-8 etc .And VEGF was considered as a very important factor involving angiogenesis, developing and fading away. The VEGF can promote angiogenesis, keeping the activation of endothelial cells, helping migration of endothelial cells, promote its proliferation, enhance the permeability of microvessel and so on. The VEGF is an very critical mitogen for endothelial cells, which can specifically work on endothelial cells to promote proliferation, migration to enhance angiogenesis. Under the condition of tumor and hypoxia or ischemia, VEGF expression and its receptors are unregulated. with the VEGF deletion, there will be lumen closure and degeneration .
By taking use of gene interference to down regulating the expression of VEGF to research and do the therapy has gain significant progress, at the same time the researchers has found that proliferating hemangioma (IH) is different from other types of blood malformation, and the earlier stage growth is caused by uncontrollable ECs, and VEGF was considered as a key factor in the process, taking VEGF as a new target for therapy to cure hemagioma is partly using in diagnosis and clinical treatment .
This research work using RNA interference technique to down regulate VEGF expression level in hemangioma mouse model and infant hemangioma ECs in vivo and in vitro respectively to investigate hemangioma and hemangioma ECs activity and development, also to detect the mechanism of hemangioma proliferation and fading away, in order to provide solid evidence for future clinical therapy.
1. The modeling of nude mice hemangioma transplantation
Aim: To establish the nude mice to mentum hemangioma transplantation model to evaluate the best condition. Method: Taking the surgery cutting estrogen receptor positive hemangioma tissue at the stage of proliferation from children to plant the tissues under the skin of 20 BALB/c nude mice, four sites on every nude mouse, and then divided these mice into four groups, group1 was fed with normal food after transplantation, group 2 was injected with estrogen 0.01 mg every week at the basis of group1, group3 was injected with estrogen 0.1mg every week at the basis of group 1, and group 4 was injected with estrogen 1mg every week, after 30,60,90 days respectively, to harvest the transplantation hemangioma and then them into pathological examination by microscope, and also did the immunohistochemistry staining by CD31, CD34,Ki-67 monoantibody to these samples. Results: At the earlier stage after transplantation there were large sums of ECs degeneration and necrosis, part of the transplantation hemangioma began to absorb and being state of fibrosis in the group1 and group2 after 30 days, and in the group3 and group4 the transplantation tissue began to proliferate gradually. After 90 days we found that there were no ECs survival in the group1 and group2, part of hemangioma survived in the group4, and in the group3, all the transplantation hemangioma survived. Under the microscope, there are similarities compare with the original hemangioma. Conclusion: Different does of estrogen has different effect on the establish of the nude mice hemangioma transplantation model, proper does estrogen can help to establish stable human hemangioma nude mice transpaltation model, which could be taken into preclinical and clinical research .
2. The culture and identification of proliferating hemangioma ECs in vitro
Aim: To culture and identify the hemangioma endothelial cell(HemEC). Methods: To collect the surgery cutting proliferating hemangioma tissue under the condition of sterile, and using the tissue to culture. By using the M199 medium with 20% FBS adding endothelial cell growth supplement (ECGS 100μg/ml) in the 5%CO2、37℃incubator, we changed the medium every 2 -3 days, and passage them until the bottle of the cell culture bottle is full. To watch the growth and morphological changes by the microscope and to identify the cells by the TEM and to test theⅧfactor associated antigen by immunohistochemistry. Results: There were cell migration from the edge of the tissue after 3 days of plantation, cells became polygon, after 1 week the cells mixed into pieces and part of the cells mix like isles .About 3 weeks later the bottle bottom was full and then passage the cells, there were typical pebble on the bottom, theⅧfactor associated antigen was positive by immunohistochemistry the TEM showed there were Weibel-Palade bodied which was the characteristic of ECs in the cells. Conclusion: By using the explants could acquire relatively pure hemangioma Ecs, and this method was easy and effective.
3. The effect of VEGF interference plasmid on hemangioma ECs
Aim: By using VEGF interference plasmid to work on the proliferating hemangioma ECs and watch the VEGF secretion and its effect. Method: Transfection of expression plasmid pGCsi-U6NeoRFP-shVEGF into HemECs, to watch the transfection efficiency by the Inverted fluorescence microscope; to exam the effect of sh VEGF to HemECs by MTT and ELISA. Results: Compared with the control group, the transfected HemECs VEGF expression lever decreased significantly, in the medium of the transfected group, the expression of VEGF in the 1st, 3rd, 5th and 7th day is 141.3±7.82、92.34±5.71、68.07±5.95 and 40.65±6.71pg/ml(P < 0.05), respectively, and the down regulation of VEGF also promoted cell apoptosis. Conclusion: Transfection of the VEGF interference plasmid could decrease the VEGF expression significantly in the HemECs and also promote apoptosis.
4. The effect of VEGF interference lentivirus on the nude mice hemangioma transplantation model
Aim: To establish the VEGF interference lentivirus Lenti-siRNA-VEGF, then work it on the nude mice hemangioma transplantation model to watch the growth and apoptosis of the hemangioma in order to discover new therapy for it. Methods: We constructed Lenti-siRNA-VEGF and established. The nude mice hemangioma transplantation model and then took Lenti-siRNA-VEGF in use on the transplantation model to watch the morphological changes, taking HE, immunohistochemistry and TUNEL to watch the hemangioma sections. By using western blotting to evaluate the growth of hemagioma and fading away statement after VEGF was down regulated. Results: The hemangioma began to became smaller after injection of Lenti-siRNA-VEGF 1 week later, and one month later the body of the hemangioma began to reduce and hardening, at the same time the control group without changes. HE staining showed that vessel space closed up and hemangioma fibrosis. CD31, CD34 immunohistochemistry showed that the positive cells were 7.68±0.83, 10.25±1.29(P<0.05) respectively. TUNEL assay found that there were more apoptosis cell in the experiment group. Western blotting showed that VEGF protein expression decreased significantly. Conclusion: The Lenti-siRNA-VEGF worked on transplantation model can down regulating the VEGF significantly, also promoted HemECs apoptosis. This method supplied an effective way for further experiment and clinical research.
引文
[1] Elsayes K M, Menias C O, Dillman J R, et al. Vascular malformation and hemangiomatosis syndromes: spectrum of imaging manifestations.[J]. 2008.
[2] Mulliken J B, Glowacki J. Hemangiomas and vascular malformations in infants and children: a classification based on endothelial characteristics.[J]. Plast Reconstr Surg, 1982, 69(3): 412-422.
[3] Coultas L, Chawengsaksophak K, Rossant J. Endothelial cells and VEGF in vascular development.[J]. Nature, 2005, 438(7070): 937-945.
[4] Otrock Z K, Makarem J A, Shamseddine A I. Vascular endothelial growth factor family of ligands and receptors: review.[J]. Blood Cells Mol Dis, 2007, 38(3): 258-268.
[5] Mei J, Gao Y, Zhang L, et al. VEGF-siRNA silencing induces apoptosis, inhibits proliferation and suppresses vasculogenic mimicry in osteosarcoma in vitro.[J]. Exp Oncol, 2008, 30(1): 29-34.
[6] Kim S H, Jeong J H, Lee S H, et al. Local and systemic delivery of VEGF siRNA using polyelectrolyte complex micelles for effective treatment of cancer.[J]. J Control Release, 2008, 129(2): 107-116.
[7] Mulliken J B, Fishman S J, Burrows P E. Vascular anomalies.[J]. Curr Probl Surg, 2000, 37(8): 517-584.
[8] Metry D W, Haggstrom A N, Drolet B A, et al. A prospective study of PHACE syndrome in infantile hemangiomas: demographic features, clinical findings, and complications.[J]. Am J Med Genet A, 2006, 140(9): 975-986.
[9] Boye E, Jinnin M, Olsen B R. Infantile hemangioma: challenges, new insights, and therapeutic promise.[J]. J Craniofac Surg, 2009, 20 Suppl 1:678-684.
[10] Lo K, Mihm M, Fay A. Current theories on the pathogenesis of infantile hemangioma.[J]. Semin Ophthalmol, 2009, 24(3): 172-177.
[11] Ritter M R, Butschek R A, Friedlander M, et al. Pathogenesis of infantile haemangioma: new molecular and cellular insights.[J]. Expert Rev Mol Med, 2007, 9(32): 1-19.
[12] Boye E, Yu Y, Paranya G, et al. Clonality and altered behavior of endothelial cells from hemangiomas.[J]. J Clin Invest, 2001, 107(6): 745-752.
[13] Walter J W, North P E, Waner M, et al. Somatic mutation of vascular endothelial growth factor receptors in juvenile hemangioma.[J]. Genes Chromosomes Cancer, 2002, 33(3): 295-303.
[14] Barnes C M, Huang S, Kaipainen A, et al. Evidence by molecular profiling for a placental origin of infantile hemangioma.[J]. Proc Natl Acad Sci U S A, 2005, 102(52): 19097-19102.
[15] Fishman S J, Mulliken J B. Hemangiomas and vascular malformations of infancy and childhood.[J]. Pediatr Clin North Am, 1993, 40(6): 1177-1200.
[16] Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor endothelial cells for angiogenesis.[J]. Science, 1997, 275(5302): 964-967.
[17] Kleinman M E, Tepper O M, Capla J M, et al. Increased circulating AC133+ CD34+ endothelial progenitor cells in children with hemangioma.[J]. Lymphat Res Biol, 2003, 1(4): 301-307.
[18] Yu Y, Flint A F, Mulliken J B, et al. Endothelial progenitor cells in infantile hemangioma.[J]. Blood, 2004, 103(4): 1373-1375.
[19] Khan Z A, Melero-Martin J M, Wu X, et al. Endothelial progenitor cells from infantile hemangioma and umbilical cord blood display unique cellular responses to endostatin.[J]. Blood, 2006, 108(3): 915-921.
[20] North P E, Mizeracki A, Mihm M J, et al. GLUT1 immunoreaction patterns reliably distinguish hemangioblastoma from metastatic renal cell carcinoma.[J]. Clin Neuropathol, 2000, 19(3): 131-137.
[21] North P E, Waner M, Mizeracki A, et al. GLUT1: a newly discovered immunohistochemical marker for juvenile hemangiomas.[J]. Hum Pathol, 2000, 31(1): 11-22.
[22] North P E, Waner M, Brodsky M C. Are infantile hemangiomas of placental origin?[J]. Ophthalmology, 2002, 109(4): 633-634.
[23] Haggstrom A N, Drolet B A, Baselga E, et al. Prospective study of infantile hemangiomas: demographic, prenatal, and perinatal characteristics.[J]. J Pediatr, 2007, 150(3): 291-294.
[24] Burton B K, Schulz C J, Angle B, et al. An increased incidence of haemangiomas in infants born following chorionic villus sampling (CVS).[J]. Prenat Diagn, 1995, 15(3): 209-214.
[25] Barnes C M, Christison-Lagay E A, Folkman J. The placenta theory and the origin of infantile hemangioma.[J]. Lymphat Res Biol, 2007, 5(4): 245-255.
[26] Mcmillan J R, Mcgrath J A, Pulkkinen L, et al. Immunohistochemical analysis of the skin in junctional epidermolysis bullosa using laminin 5 chain specific antibodies is of limited value in predicting the underlying gene mutation.[J]. Br J Dermatol, 1997, 136(6): 817-822.
[27] Pasyk K A, Argenta L C, Austad E D. Histopathology of human expanded tissue.[J]. Clin Plast Surg, 1987, 14(3): 435-445.
[28] Tan S T, Hasan Q, Velickovic M, et al. A novel in vitro human model of hemangioma.[J]. Mod Pathol, 2000, 13(1): 92-99.
[29] Dadras S S, North P E, Bertoncini J, et al. Infantile hemangiomas are arrested in an early developmental vascular differentiation state.[J]. Mod Pathol,2004, 17(9): 1068-1079.
[30] Cejudo-Martin P, Johnson R S. A new notch in the HIF belt: how hypoxia impacts differentiation.[J]. Dev Cell, 2005, 9(5): 575-576.
[31] Ceradini D J, Kulkarni A R, Callaghan M J, et al. Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1 induction of SDF-1.[J]. Nat Med, 2004, 10(8): 858-864.
[32] Manalo D J, Rowan A, Lavoie T, et al. Transcriptional regulation of vascular endothelial cell responses to hypoxia by HIF-1.[J]. Blood, 2005, 105(2): 659-669.
[33] Takahashi K, Mulliken J B, Kozakewich H P, et al. Cellular markers that distinguish the phases of hemangioma during infancy and childhood.[J]. J Clin Invest, 1994, 93(6): 2357-2364.
[34] Yu Y, Varughese J, Brown L F, et al. Increased Tie2 expression, enhanced response to angiopoietin-1, and dysregulated angiopoietin-2 expression in hemangioma-derived endothelial cells.[J]. Am J Pathol, 2001, 159(6): 2271-2280.
[35] Bielenberg D R, Bucana C D, 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.[J]. Int J Oncol, 1999, 14(3): 401-408.
[36] Feldser D, Agani F, Iyer N V, et al. Reciprocal positive regulation of hypoxia-inducible factor 1alpha and insulin-like growth factor 2.[J]. Cancer Res, 1999, 59(16): 3915-3918.
[37] Razandi M, Pedram A, Levin E R. Estrogen signals to the preservation of endothelial cell form and function.[J]. J Biol Chem, 2000, 275(49):38540-38546.
[38] Xiao X, Hong L, Sheng M. Promoting effect of estrogen on the proliferation of hemangioma vascular endothelial cells in vitro.[J]. J Pediatr Surg, 1999, 34(11): 1603-1605.
[39] Kleinman M E, Greives M R, Churgin S S, et al. Hypoxia-induced mediators of stem/progenitor cell trafficking are increased in children with hemangioma.[J]. Arterioscler Thromb Vasc Biol, 2007, 27(12): 2664-2670.
[40] Heissig B, Rafii S, Akiyama H, et al. Low-dose irradiation promotes tissue revascularization through VEGF release from mast cells and MMP-9-mediated progenitor cell mobilization.[J]. J Exp Med, 2005, 202(6): 739-750.
[41] Sun Z Y, Yang L, Yi C G, et al. Possibilities and potential roles of estrogen in the pathogenesis of proliferation hemangiomas formation.[J]. Med Hypotheses, 2008, 71(2): 286-292.
[42] Razon M J, Kraling B M, Mulliken J B, et al. Increased apoptosis coincides with onset of involution in infantile hemangioma.[J]. Microcirculation, 1998, 5(2-3): 189-195.
[43] Halperin R, Peller S, Rotschild M, et al. Placental apoptosis in normal and abnormal pregnancies.[J]. Gynecol Obstet Invest, 2000, 50(2): 84-87.
[44] Yu Y, Wylie-Sears J, Boscolo E, et al. Genomic imprinting of IGF2 is maintained in infantile hemangioma despite its high level of expression.[J]. Mol Med, 2004, 10(7-12): 117-123.
[45] Ritter M R, Moreno S K, Dorrell M I, et al. Identifying potential regulators of infantile hemangioma progression through large-scale expression analysis: a possible role for the immune system and indoleamine 2,3 dioxygenase (IDO) during involution.[J]. Lymphat Res Biol, 2003, 1(4): 291-299.
[46] Mellor A L, Sivakumar J, Chandler P, et al. Prevention of T cell-drivencomplement activation and inflammation by tryptophan catabolism during pregnancy.[J]. Nat Immunol, 2001, 2(1): 64-68.
[47] Tan S T, Wallis R A, He Y, et al. Mast cells and hemangioma.[J]. Plast Reconstr Surg, 2004, 113(3): 999-1011.
[48] Hasan Q, Ruger B M, Tan S T, et al. Clusterin/apoJ expression during the development of hemangioma.[J]. Hum Pathol, 2000, 31(6): 691-697.
[49] Schlunt L B, Harper J D, Broome D R, et al. Improved detection of renal vascular anatomy using multidetector CT angiography: Is 100% detection possible?[J]. J Endourol, 2007, 21(1): 12-17.
[50] Zarem H A, Edgerton M T. Induced resolution of cavernous hemangiomas following prednisolone therapy.[J]. Plast Reconstr Surg, 1967, 39(1): 76-83.
[51] Ebrahem Q, Minamoto A, Hoppe G, et al. Triamcinolone acetonide inhibits IL-6- and VEGF-induced angiogenesis downstream of the IL-6 and VEGF receptors.[J]. Invest Ophthalmol Vis Sci, 2006, 47(11): 4935-4941.
[52] Ezekowitz R A, Mulliken J B, Folkman J. Interferon alfa-2a therapy for life-threatening hemangiomas of infancy.[J]. N Engl J Med, 1992, 326(22): 1456-1463.
[53] Michaud A P, Bauman N M, Burke D K, et al. Spastic diplegia and other motor disturbances in infants receiving interferon-alpha.[J]. Laryngoscope, 2004, 114(7): 1231-1236.
[54] Sanchez C I, Mihm M C, Waner M. [Laser and intense pulsed light in the treatment of infantile haemangiomas and vascular malformations][J]. An Sist Sanit Navar, 2004, 27 Suppl 1: 103-115.
[55] Sans V, Dumas D L R E, Berge J, et al. Propranolol for Severe Infantile Hemangiomas: Follow-Up Report.[J]. Pediatrics, 2009.
[56] Senger D R, Galli S J, Dvorak A M, et al. Tumor cells secrete a vascularpermeability factor that promotes accumulation of ascites fluid.[J]. Science, 1983, 219(4587): 983-985.
[57] Connolly D T, Heuvelman D M, Nelson R, et al. Tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis.[J]. J Clin Invest, 1989, 84(5): 1470-1478.
[58] Rini B I, Small E J. Biology and clinical development of vascular endothelial growth factor-targeted therapy in renal cell carcinoma.[J]. J Clin Oncol, 2005, 23(5): 1028-1043.
[59] Tischer E, Mitchell R, Hartman T, et al. The human gene for vascular endothelial growth factor. Multiple protein forms are encoded through alternative exon splicing.[J]. J Biol Chem, 1991, 266(18): 11947-11954.
[60] Sheen I S, Jeng K S, Shih S C, et al. Clinical significance of the expression of isoform 165 vascular endothelial growth factor mRNA in noncancerous liver remnants of patients with hepatocellular carcinoma.[J]. World J Gastroenterol, 2005, 11(2): 187-192.
[61] Carmeliet P, Ferreira V, Breier G, et al. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele.[J]. Nature, 1996, 380(6573): 435-439.
[62] Xia Y P, Li B, Hylton D, et al. Transgenic delivery of VEGF to mouse skin leads to an inflammatory condition resembling human psoriasis.[J]. Blood, 2003, 102(1): 161-168.
[63] Ferrara N, Gerber H P, Lecouter J. The biology of VEGF and its receptors.[J]. Nat Med, 2003, 9(6): 669-676.
[64] Ackah E, Yu J, Zoellner S, et al. Akt1/protein kinase Balpha is critical for ischemic and VEGF-mediated angiogenesis.[J]. J Clin Invest, 2005, 115(8): 2119-2127.
[65] Chang J, Most D, Bresnick S, et al. Proliferative hemangiomas: analysis of cytokine gene expression and angiogenesis.[J]. Plast Reconstr Surg, 1999, 103(1): 1-9, 10.
[66] Jinnin M, Medici D, Park L, et al. Suppressed NFAT-dependent VEGFR1 expression and constitutive VEGFR2 signaling in infantile hemangioma.[J]. Nat Med, 2008, 14(11): 1236-1246.
[67] 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.[J]. Am J Pathol, 1997, 150(4): 1315-1326.
[68] Boscolo E, Bischoff J. Vasculogenesis in infantile hemangioma.[J]. Angiogenesis, 2009, 12(2): 197-207.
[69] Zhang L, Lin X, Wang W, et al. Circulating level of vascular endothelial growth factor in differentiating hemangioma from vascular malformation patients.[J]. Plast Reconstr Surg, 2005, 116(1): 200-204.
[70] Kitajima S, Liu E, Morimoto M, et al. Transgenic rabbits with increased VEGF expression develop hemangiomas in the liver: a new model for Kasabach-Merritt syndrome.[J]. Lab Invest, 2005, 85(12): 1517-1527.
[71] Ozawa C R, Banfi A, Glazer N L, et al. Microenvironmental VEGF concentration, not total dose, determines a threshold between normal and aberrant angiogenesis.[J]. J Clin Invest, 2004, 113(4): 516-527.
[72] Chen S F, Fei X, Li S H. A new simple method for isolation of microvascular endothelial cells avoiding both chemical and mechanical injuries.[J]. Microvasc Res, 1995, 50(1): 119-128.
[73] Vailhe B, Vittet D, Feige J J. In vitro models of vasculogenesis and angiogenesis.[J]. Lab Invest, 2001, 81(4): 439-452.
[74] Dubois N A, Kolpack L C, Wang R, et al. Isolation and characterization ofan established endothelial cell line from transgenic mouse hemangiomas.[J]. Exp Cell Res, 1991, 196(2): 302-313.
[75] Soldatskii I, Shekhter A B, Ponkratenko A D, et al. [Effect of cryotherapy and laser destruction on experimental model of human vascular tumor][J]. Vestn Otorinolaringol, 1995(2): 10-14.
[76] Peng Q, Liu W, Tang Y, et al. The establishment of the hemangioma model in nude mouse.[J]. J Pediatr Surg, 2005, 40(7): 1167-1172.
[77] Wang R, Bautch V L. The polyomavirus early region gene in transgenic mice causes vascular and bone tumors.[J]. J Virol, 1991, 65(10): 5174-5183.
[78] Wang Y A, Zheng J W, Fei Z L, et al. A novel transgenic mice model for venous malformation.[J]. Transgenic Res, 2009, 18(2): 193-201.
[79]徐骎,张志愿,陈万涛,等.转基因小鼠血管瘤动物模型建立[J].中华口腔医学杂志, 2003, 38(5): 355-357.
[80]刘军,宋健星,邢新,等.血管内皮细胞生长因子基因移植制备大鼠血管瘤模型[J].第二军医大学学报, 2005, 26(10): 1124-1127.
[81] Khan Z A, Boscolo E, Picard A, et al. Multipotential stem cells recapitulate human infantile hemangioma in immunodeficient mice.[J]. J Clin Invest, 2008, 118(7): 2592-2599.
[82]虎小毅,杨壮群,宋勇,等.细胞悬液接种法血管瘤裸鼠动物模型的建立[J].中国美容医学, 2008, 17(7): 1012-1014.
[83] Grunweller A, Hartmann R K. RNA interference as a gene-specific approach for molecular medicine.[J]. Curr Med Chem, 2005, 12(26): 3143-3161.
[84] Fire A, Xu S, Montgomery M K, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.[J]. Nature, 1998, 391(6669): 806-811.
[85] Wu S Y, Mcmillan N A. Lipidic systems for in vivo siRNA delivery.[J]. AAPS J, 2009, 11(4): 639-652.
[86] Tepper O M, Mehrara B J. Gene therapy in plastic surgery.[J]. Plast Reconstr Surg, 2002, 109(2): 716-734.
[87] Gilbert R, Liu A, Petrof B, et al. Improved performance of a fully gutted adenovirus vector containing two full-length dystrophin cDNAs regulated by a strong promoter.[J]. Mol Ther, 2002, 6(4): 501-509.
[88] Kennedy P G. Potential use of herpes simplex virus (HSV) vectors for gene therapy of neurological disorders.[J]. Brain, 1997, 120 ( Pt 7): 1245-1259.
[89] White K, Nicklin S A, Baker A H. Novel vectors for in vivo gene delivery to vascular tissue.[J]. Expert Opin Biol Ther, 2007, 7(6): 809-821.
[90] Tang Y, Liu W, Yu S, et al. A novel in vivo model of human hemangioma: xenograft of human hemangioma tissue on nude mice.[J]. Plast Reconstr Surg, 2007, 120(4): 869-878.
[91]程立新,汤少明,罗少军.血管瘤组织中雌激素、雌激素受体表达及临床意义[J].中华整形外科杂志, 2003, 19(1): 42-43.
[92] Liu J, Wu S, Wei H, et al. Effects of sex hormones and their balance on the proliferation of rat vascular endothelial cells.[J]. Horm Res, 2002, 58(1): 16-20.
[93]马戈甲,易成刚,孙智勇,等.不同剂量雌激素对裸鼠血管瘤模型的影响[J].中国美容医学, 2010, 19(2): 200-203.
[94] Tseng Y C, Mozumdar S, Huang L. Lipid-based systemic delivery of siRNA.[J]. Adv Drug Deliv Rev, 2009, 61(9): 721-731.
[2] Mulliken J B, Glowacki J. Hemangiomas and vascular malformations in infants and children: a classification based on endothelial characteristics.[J]. Plast Reconstr Surg, 1982, 69(3): 412-422.
[3] Coultas L, Chawengsaksophak K, Rossant J. Endothelial cells and VEGF in vascular development.[J]. Nature, 2005, 438(7070): 937-945.
[4] Otrock Z K, Makarem J A, Shamseddine A I. Vascular endothelial growth factor family of ligands and receptors: review.[J]. Blood Cells Mol Dis, 2007, 38(3): 258-268.
[5] Mei J, Gao Y, Zhang L, et al. VEGF-siRNA silencing induces apoptosis, inhibits proliferation and suppresses vasculogenic mimicry in osteosarcoma in vitro.[J]. Exp Oncol, 2008, 30(1): 29-34.
[6] Kim S H, Jeong J H, Lee S H, et al. Local and systemic delivery of VEGF siRNA using polyelectrolyte complex micelles for effective treatment of cancer.[J]. J Control Release, 2008, 129(2): 107-116.
[7] Mulliken J B, Fishman S J, Burrows P E. Vascular anomalies.[J]. Curr Probl Surg, 2000, 37(8): 517-584.
[8] Metry D W, Haggstrom A N, Drolet B A, et al. A prospective study of PHACE syndrome in infantile hemangiomas: demographic features, clinical findings, and complications.[J]. Am J Med Genet A, 2006, 140(9): 975-986.
[9] Boye E, Jinnin M, Olsen B R. Infantile hemangioma: challenges, new insights, and therapeutic promise.[J]. J Craniofac Surg, 2009, 20 Suppl 1:678-684.
[10] Lo K, Mihm M, Fay A. Current theories on the pathogenesis of infantile hemangioma.[J]. Semin Ophthalmol, 2009, 24(3): 172-177.
[11] Ritter M R, Butschek R A, Friedlander M, et al. Pathogenesis of infantile haemangioma: new molecular and cellular insights.[J]. Expert Rev Mol Med, 2007, 9(32): 1-19.
[12] Boye E, Yu Y, Paranya G, et al. Clonality and altered behavior of endothelial cells from hemangiomas.[J]. J Clin Invest, 2001, 107(6): 745-752.
[13] Walter J W, North P E, Waner M, et al. Somatic mutation of vascular endothelial growth factor receptors in juvenile hemangioma.[J]. Genes Chromosomes Cancer, 2002, 33(3): 295-303.
[14] Barnes C M, Huang S, Kaipainen A, et al. Evidence by molecular profiling for a placental origin of infantile hemangioma.[J]. Proc Natl Acad Sci U S A, 2005, 102(52): 19097-19102.
[15] Fishman S J, Mulliken J B. Hemangiomas and vascular malformations of infancy and childhood.[J]. Pediatr Clin North Am, 1993, 40(6): 1177-1200.
[16] Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor endothelial cells for angiogenesis.[J]. Science, 1997, 275(5302): 964-967.
[17] Kleinman M E, Tepper O M, Capla J M, et al. Increased circulating AC133+ CD34+ endothelial progenitor cells in children with hemangioma.[J]. Lymphat Res Biol, 2003, 1(4): 301-307.
[18] Yu Y, Flint A F, Mulliken J B, et al. Endothelial progenitor cells in infantile hemangioma.[J]. Blood, 2004, 103(4): 1373-1375.
[19] Khan Z A, Melero-Martin J M, Wu X, et al. Endothelial progenitor cells from infantile hemangioma and umbilical cord blood display unique cellular responses to endostatin.[J]. Blood, 2006, 108(3): 915-921.
[20] North P E, Mizeracki A, Mihm M J, et al. GLUT1 immunoreaction patterns reliably distinguish hemangioblastoma from metastatic renal cell carcinoma.[J]. Clin Neuropathol, 2000, 19(3): 131-137.
[21] North P E, Waner M, Mizeracki A, et al. GLUT1: a newly discovered immunohistochemical marker for juvenile hemangiomas.[J]. Hum Pathol, 2000, 31(1): 11-22.
[22] North P E, Waner M, Brodsky M C. Are infantile hemangiomas of placental origin?[J]. Ophthalmology, 2002, 109(4): 633-634.
[23] Haggstrom A N, Drolet B A, Baselga E, et al. Prospective study of infantile hemangiomas: demographic, prenatal, and perinatal characteristics.[J]. J Pediatr, 2007, 150(3): 291-294.
[24] Burton B K, Schulz C J, Angle B, et al. An increased incidence of haemangiomas in infants born following chorionic villus sampling (CVS).[J]. Prenat Diagn, 1995, 15(3): 209-214.
[25] Barnes C M, Christison-Lagay E A, Folkman J. The placenta theory and the origin of infantile hemangioma.[J]. Lymphat Res Biol, 2007, 5(4): 245-255.
[26] Mcmillan J R, Mcgrath J A, Pulkkinen L, et al. Immunohistochemical analysis of the skin in junctional epidermolysis bullosa using laminin 5 chain specific antibodies is of limited value in predicting the underlying gene mutation.[J]. Br J Dermatol, 1997, 136(6): 817-822.
[27] Pasyk K A, Argenta L C, Austad E D. Histopathology of human expanded tissue.[J]. Clin Plast Surg, 1987, 14(3): 435-445.
[28] Tan S T, Hasan Q, Velickovic M, et al. A novel in vitro human model of hemangioma.[J]. Mod Pathol, 2000, 13(1): 92-99.
[29] Dadras S S, North P E, Bertoncini J, et al. Infantile hemangiomas are arrested in an early developmental vascular differentiation state.[J]. Mod Pathol,2004, 17(9): 1068-1079.
[30] Cejudo-Martin P, Johnson R S. A new notch in the HIF belt: how hypoxia impacts differentiation.[J]. Dev Cell, 2005, 9(5): 575-576.
[31] Ceradini D J, Kulkarni A R, Callaghan M J, et al. Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1 induction of SDF-1.[J]. Nat Med, 2004, 10(8): 858-864.
[32] Manalo D J, Rowan A, Lavoie T, et al. Transcriptional regulation of vascular endothelial cell responses to hypoxia by HIF-1.[J]. Blood, 2005, 105(2): 659-669.
[33] Takahashi K, Mulliken J B, Kozakewich H P, et al. Cellular markers that distinguish the phases of hemangioma during infancy and childhood.[J]. J Clin Invest, 1994, 93(6): 2357-2364.
[34] Yu Y, Varughese J, Brown L F, et al. Increased Tie2 expression, enhanced response to angiopoietin-1, and dysregulated angiopoietin-2 expression in hemangioma-derived endothelial cells.[J]. Am J Pathol, 2001, 159(6): 2271-2280.
[35] Bielenberg D R, Bucana C D, 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.[J]. Int J Oncol, 1999, 14(3): 401-408.
[36] Feldser D, Agani F, Iyer N V, et al. Reciprocal positive regulation of hypoxia-inducible factor 1alpha and insulin-like growth factor 2.[J]. Cancer Res, 1999, 59(16): 3915-3918.
[37] Razandi M, Pedram A, Levin E R. Estrogen signals to the preservation of endothelial cell form and function.[J]. J Biol Chem, 2000, 275(49):38540-38546.
[38] Xiao X, Hong L, Sheng M. Promoting effect of estrogen on the proliferation of hemangioma vascular endothelial cells in vitro.[J]. J Pediatr Surg, 1999, 34(11): 1603-1605.
[39] Kleinman M E, Greives M R, Churgin S S, et al. Hypoxia-induced mediators of stem/progenitor cell trafficking are increased in children with hemangioma.[J]. Arterioscler Thromb Vasc Biol, 2007, 27(12): 2664-2670.
[40] Heissig B, Rafii S, Akiyama H, et al. Low-dose irradiation promotes tissue revascularization through VEGF release from mast cells and MMP-9-mediated progenitor cell mobilization.[J]. J Exp Med, 2005, 202(6): 739-750.
[41] Sun Z Y, Yang L, Yi C G, et al. Possibilities and potential roles of estrogen in the pathogenesis of proliferation hemangiomas formation.[J]. Med Hypotheses, 2008, 71(2): 286-292.
[42] Razon M J, Kraling B M, Mulliken J B, et al. Increased apoptosis coincides with onset of involution in infantile hemangioma.[J]. Microcirculation, 1998, 5(2-3): 189-195.
[43] Halperin R, Peller S, Rotschild M, et al. Placental apoptosis in normal and abnormal pregnancies.[J]. Gynecol Obstet Invest, 2000, 50(2): 84-87.
[44] Yu Y, Wylie-Sears J, Boscolo E, et al. Genomic imprinting of IGF2 is maintained in infantile hemangioma despite its high level of expression.[J]. Mol Med, 2004, 10(7-12): 117-123.
[45] Ritter M R, Moreno S K, Dorrell M I, et al. Identifying potential regulators of infantile hemangioma progression through large-scale expression analysis: a possible role for the immune system and indoleamine 2,3 dioxygenase (IDO) during involution.[J]. Lymphat Res Biol, 2003, 1(4): 291-299.
[46] Mellor A L, Sivakumar J, Chandler P, et al. Prevention of T cell-drivencomplement activation and inflammation by tryptophan catabolism during pregnancy.[J]. Nat Immunol, 2001, 2(1): 64-68.
[47] Tan S T, Wallis R A, He Y, et al. Mast cells and hemangioma.[J]. Plast Reconstr Surg, 2004, 113(3): 999-1011.
[48] Hasan Q, Ruger B M, Tan S T, et al. Clusterin/apoJ expression during the development of hemangioma.[J]. Hum Pathol, 2000, 31(6): 691-697.
[49] Schlunt L B, Harper J D, Broome D R, et al. Improved detection of renal vascular anatomy using multidetector CT angiography: Is 100% detection possible?[J]. J Endourol, 2007, 21(1): 12-17.
[50] Zarem H A, Edgerton M T. Induced resolution of cavernous hemangiomas following prednisolone therapy.[J]. Plast Reconstr Surg, 1967, 39(1): 76-83.
[51] Ebrahem Q, Minamoto A, Hoppe G, et al. Triamcinolone acetonide inhibits IL-6- and VEGF-induced angiogenesis downstream of the IL-6 and VEGF receptors.[J]. Invest Ophthalmol Vis Sci, 2006, 47(11): 4935-4941.
[52] Ezekowitz R A, Mulliken J B, Folkman J. Interferon alfa-2a therapy for life-threatening hemangiomas of infancy.[J]. N Engl J Med, 1992, 326(22): 1456-1463.
[53] Michaud A P, Bauman N M, Burke D K, et al. Spastic diplegia and other motor disturbances in infants receiving interferon-alpha.[J]. Laryngoscope, 2004, 114(7): 1231-1236.
[54] Sanchez C I, Mihm M C, Waner M. [Laser and intense pulsed light in the treatment of infantile haemangiomas and vascular malformations][J]. An Sist Sanit Navar, 2004, 27 Suppl 1: 103-115.
[55] Sans V, Dumas D L R E, Berge J, et al. Propranolol for Severe Infantile Hemangiomas: Follow-Up Report.[J]. Pediatrics, 2009.
[56] Senger D R, Galli S J, Dvorak A M, et al. Tumor cells secrete a vascularpermeability factor that promotes accumulation of ascites fluid.[J]. Science, 1983, 219(4587): 983-985.
[57] Connolly D T, Heuvelman D M, Nelson R, et al. Tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis.[J]. J Clin Invest, 1989, 84(5): 1470-1478.
[58] Rini B I, Small E J. Biology and clinical development of vascular endothelial growth factor-targeted therapy in renal cell carcinoma.[J]. J Clin Oncol, 2005, 23(5): 1028-1043.
[59] Tischer E, Mitchell R, Hartman T, et al. The human gene for vascular endothelial growth factor. Multiple protein forms are encoded through alternative exon splicing.[J]. J Biol Chem, 1991, 266(18): 11947-11954.
[60] Sheen I S, Jeng K S, Shih S C, et al. Clinical significance of the expression of isoform 165 vascular endothelial growth factor mRNA in noncancerous liver remnants of patients with hepatocellular carcinoma.[J]. World J Gastroenterol, 2005, 11(2): 187-192.
[61] Carmeliet P, Ferreira V, Breier G, et al. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele.[J]. Nature, 1996, 380(6573): 435-439.
[62] Xia Y P, Li B, Hylton D, et al. Transgenic delivery of VEGF to mouse skin leads to an inflammatory condition resembling human psoriasis.[J]. Blood, 2003, 102(1): 161-168.
[63] Ferrara N, Gerber H P, Lecouter J. The biology of VEGF and its receptors.[J]. Nat Med, 2003, 9(6): 669-676.
[64] Ackah E, Yu J, Zoellner S, et al. Akt1/protein kinase Balpha is critical for ischemic and VEGF-mediated angiogenesis.[J]. J Clin Invest, 2005, 115(8): 2119-2127.
[65] Chang J, Most D, Bresnick S, et al. Proliferative hemangiomas: analysis of cytokine gene expression and angiogenesis.[J]. Plast Reconstr Surg, 1999, 103(1): 1-9, 10.
[66] Jinnin M, Medici D, Park L, et al. Suppressed NFAT-dependent VEGFR1 expression and constitutive VEGFR2 signaling in infantile hemangioma.[J]. Nat Med, 2008, 14(11): 1236-1246.
[67] 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.[J]. Am J Pathol, 1997, 150(4): 1315-1326.
[68] Boscolo E, Bischoff J. Vasculogenesis in infantile hemangioma.[J]. Angiogenesis, 2009, 12(2): 197-207.
[69] Zhang L, Lin X, Wang W, et al. Circulating level of vascular endothelial growth factor in differentiating hemangioma from vascular malformation patients.[J]. Plast Reconstr Surg, 2005, 116(1): 200-204.
[70] Kitajima S, Liu E, Morimoto M, et al. Transgenic rabbits with increased VEGF expression develop hemangiomas in the liver: a new model for Kasabach-Merritt syndrome.[J]. Lab Invest, 2005, 85(12): 1517-1527.
[71] Ozawa C R, Banfi A, Glazer N L, et al. Microenvironmental VEGF concentration, not total dose, determines a threshold between normal and aberrant angiogenesis.[J]. J Clin Invest, 2004, 113(4): 516-527.
[72] Chen S F, Fei X, Li S H. A new simple method for isolation of microvascular endothelial cells avoiding both chemical and mechanical injuries.[J]. Microvasc Res, 1995, 50(1): 119-128.
[73] Vailhe B, Vittet D, Feige J J. In vitro models of vasculogenesis and angiogenesis.[J]. Lab Invest, 2001, 81(4): 439-452.
[74] Dubois N A, Kolpack L C, Wang R, et al. Isolation and characterization ofan established endothelial cell line from transgenic mouse hemangiomas.[J]. Exp Cell Res, 1991, 196(2): 302-313.
[75] Soldatskii I, Shekhter A B, Ponkratenko A D, et al. [Effect of cryotherapy and laser destruction on experimental model of human vascular tumor][J]. Vestn Otorinolaringol, 1995(2): 10-14.
[76] Peng Q, Liu W, Tang Y, et al. The establishment of the hemangioma model in nude mouse.[J]. J Pediatr Surg, 2005, 40(7): 1167-1172.
[77] Wang R, Bautch V L. The polyomavirus early region gene in transgenic mice causes vascular and bone tumors.[J]. J Virol, 1991, 65(10): 5174-5183.
[78] Wang Y A, Zheng J W, Fei Z L, et al. A novel transgenic mice model for venous malformation.[J]. Transgenic Res, 2009, 18(2): 193-201.
[79]徐骎,张志愿,陈万涛,等.转基因小鼠血管瘤动物模型建立[J].中华口腔医学杂志, 2003, 38(5): 355-357.
[80]刘军,宋健星,邢新,等.血管内皮细胞生长因子基因移植制备大鼠血管瘤模型[J].第二军医大学学报, 2005, 26(10): 1124-1127.
[81] Khan Z A, Boscolo E, Picard A, et al. Multipotential stem cells recapitulate human infantile hemangioma in immunodeficient mice.[J]. J Clin Invest, 2008, 118(7): 2592-2599.
[82]虎小毅,杨壮群,宋勇,等.细胞悬液接种法血管瘤裸鼠动物模型的建立[J].中国美容医学, 2008, 17(7): 1012-1014.
[83] Grunweller A, Hartmann R K. RNA interference as a gene-specific approach for molecular medicine.[J]. Curr Med Chem, 2005, 12(26): 3143-3161.
[84] Fire A, Xu S, Montgomery M K, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.[J]. Nature, 1998, 391(6669): 806-811.
[85] Wu S Y, Mcmillan N A. Lipidic systems for in vivo siRNA delivery.[J]. AAPS J, 2009, 11(4): 639-652.
[86] Tepper O M, Mehrara B J. Gene therapy in plastic surgery.[J]. Plast Reconstr Surg, 2002, 109(2): 716-734.
[87] Gilbert R, Liu A, Petrof B, et al. Improved performance of a fully gutted adenovirus vector containing two full-length dystrophin cDNAs regulated by a strong promoter.[J]. Mol Ther, 2002, 6(4): 501-509.
[88] Kennedy P G. Potential use of herpes simplex virus (HSV) vectors for gene therapy of neurological disorders.[J]. Brain, 1997, 120 ( Pt 7): 1245-1259.
[89] White K, Nicklin S A, Baker A H. Novel vectors for in vivo gene delivery to vascular tissue.[J]. Expert Opin Biol Ther, 2007, 7(6): 809-821.
[90] Tang Y, Liu W, Yu S, et al. A novel in vivo model of human hemangioma: xenograft of human hemangioma tissue on nude mice.[J]. Plast Reconstr Surg, 2007, 120(4): 869-878.
[91]程立新,汤少明,罗少军.血管瘤组织中雌激素、雌激素受体表达及临床意义[J].中华整形外科杂志, 2003, 19(1): 42-43.
[92] Liu J, Wu S, Wei H, et al. Effects of sex hormones and their balance on the proliferation of rat vascular endothelial cells.[J]. Horm Res, 2002, 58(1): 16-20.
[93]马戈甲,易成刚,孙智勇,等.不同剂量雌激素对裸鼠血管瘤模型的影响[J].中国美容医学, 2010, 19(2): 200-203.
[94] Tseng Y C, Mozumdar S, Huang L. Lipid-based systemic delivery of siRNA.[J]. Adv Drug Deliv Rev, 2009, 61(9): 721-731.