Bcl-2、Bax、Fas和Caspase-3在血管瘤中的表达及意义
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摘要
目的：血管瘤是最常见的良性肿瘤之一，在婴幼儿中发病率较高。在以往的临床诊断中，血管瘤的命名和分类比较混乱，往往不加区分的将“血管瘤”一词用于多种血管性疾病，包括血管畸形。血管瘤是由内皮细胞大量增殖而形成的良性肿瘤，其内皮细胞分裂率异常增高，并且有自发退化的倾向；血管畸形是胚胎血管发生过程中的结构异常，其内皮细胞分裂率正常，而且血管畸形不会自发的退化。根据血管瘤内皮细胞的生物学特点并结合临床，将血管瘤的自然病程分为增生期、退化期和退化完成期。目前，血管瘤的发生、发展和退化机制尚未完全明了。现有的研究发现，内皮细胞的增殖状态的改变、多种血管形成因子和血管形成抑制因子在血管瘤不同时期表达水平的变化以及内皮细胞增殖与凋亡的不平衡与血管瘤的发生、发展和退化有密切关系。目前的实验研究中，尤其引人关注的焦点的是血管瘤自发消退的机制。已有研究发现，在血管瘤组织中存在着较高的细胞凋亡率，而血管畸形中则没有，那么为什么血管瘤可以自发消退而血管畸形则不能，其机制是否与细胞凋亡有关?Bcl-2、Bax、Fas和Caspase-3是在细胞凋亡发生过程中具有比较重要地位和作用的蛋白质，也是近年来细胞凋亡分子机制研究领域的热点。目前，Bcl-2、Bax、Fas和Caspase-3在恶性肿瘤中的研究较多，而在良性肿瘤中的研究尚不多见，在血管瘤中的研究国内外尚未见报道。为进一步探讨Bcl-2、Bax、Fas和Caspase-3与血管瘤发生、发展的关系及其分子机制，本课题应用免疫组织化学技术检测了增生期、退化期血管瘤组织和正常皮肤组织血管中Bcl-2、Bax、Fas和Caspase-3的表达。
方法：收集武汉大学人民医院病理科1996-2001年皮肤血管瘤存档蜡块49例，其中男性26例，女性23例，最小年龄2月，最大年龄68岁，平均年龄30.3岁。蜡块切片厚5μm，常规HE染色和免疫组织化学S-P法检测增殖细胞核抗原(proliferating cell nuclear antigen，PCNA)，按Mulliken分类标准并结合PCNA的表达进行分组，增生期血管瘤27例，退化期血管瘤22例，另取瘤组织周围正常皮肤组织5例作对照。运用免疫组织化学染色方法检测Bcl-2、Bax、Fas和Caspase-3在以上各组中的表达，并结合Ⅷ因子相关抗原的免疫组织化学染色证实表达Bcl-2、Bax、Fas和Caspase-3的
这些细胞是血管瘤组织中的内皮细胞。利用HPIAS一2000图像分析系统测定Bcl一2、
Bax、Fas和Caspase一3在以上三组中表达的平均光密度和平均阳性面积率。
结果:Bcl一2在增生期血管瘤内皮细胞的表达明显高于退化期血管瘤内皮细胞和正
常皮肤组织血管内皮细胞(尸<0.01);Bcl一2在退化期血管瘤内皮细胞的表达与正常皮
肤组织血管内皮细胞相比，差异无显著性(尸>0.05)。Bax、Fas和easpase一3在退化
期血管瘤内皮细胞的表达明显高于增生期血管瘤内皮细胞和正常皮肤组织血管内皮细
胞(尸<0.01);Bax和Fas在增生期血管瘤内皮细胞的表达高于正常皮肤组织血管内
皮细胞(尸<0.05);Caspase一3在增生期血管瘤内皮细胞的表达与正常皮肤组织血管内
皮细胞相比，差异无显著性(P>0.05)。
结论:Bcl一2、Bax、Fas和caspase一3参与了血管瘤的发生、发展和退化。Bcl一2通
过抑制内皮细胞凋亡而促进血管瘤的增生。Bax、Fas和Caspase一3通过诱导内皮细胞
凋亡而促进血管瘤由增生向退化的转变。
Objectives: Hemangioma, which is one of the commonest benign tumors, often occurs in infants and children. But the nomenclature and classification of hemangioma is confused in clinical diagnose before. Doctors always used 'hemangioma' to define various vascular diseases, including vascular malformations. Hemangioma is a kind of benign tumor formed by mass proliferative endothelial cells. The endothelial cell division of hemangioma increases aberrantly. And hemangioma has the tendency to involute spontaneously. Vascular malformation is a kind of structural abnormality during embryonal vasculogenesis. The endothelial cell division of vascular malformation is normal. And vascular malformation can not involute spontaneously. Based on the clinical observation and biological characters of the endothelial cells, the course of hemangioma can be divided into three phases: the proliferating, involuting and involuted phases. The mechanism of development and involution of hemangioma has not been clearly elucidated so fa
r. It has been pointed out that the development and involution of hemangioma are related to the proliferating status of hemangioma endothelium, the expression levels of various angiogenesis cytokines and angiogenesis inhibitors, and the imbalance of endothelial proliferation and apoptosis. The reasons for the spontaneous involution of hemangioma have raised more interest of researchers. The facts of much higher occurrence of apoptosis cells in hemangiomas than that in vascular malformations might provide some clues to explain the spontaneous involution of hemangiomas. Bcl-2, Bax, Fas and Caspase-3, which are hotspots in the researches on malignant tumorigenesis, have been reported to play important roles in apoptosis. As apoptosis is a crucial phenomenon in the course of hemangioma, we doubted whether these four proteins are also contributing to the developments and involution of hemangiomas. The study present here in showed the different expression patterns of Bcl-2, Bax, Fas and Caspase-3 in proliferating,
involuting hemangiomas and tissues from normal skin.
Methods: Specimens from 49 cases (26 males, and 23 females) of dermal hemangioma from the Department of Pathology in Renmin Hospital of Wuhan University from 1996 to 2001 were collected. The patients are from 2 months to 68 years old, with average age 30.3
iii
years old. The specimens were generally fixed by formalin, and embedded in paraffin. Sections of 5m thickness were made and attached to poly-L-lysine-coated glass slides. Hematoxylin and eosin (HE) staining were carried out routinely to identify the histological characters of hemangiomas. The phases of hemangiomas were identified by detecting the expression of proliferating cell nuclear antigen (PCNA) by immunohistochemistry. Based on Mulliken's standard, the hemangioma specimens were divided into two groups: proliferating hemangiomas (27 cases) and involuting hemangiomas (22 cases). Normal skin tissues around hemangiomas from 5 cases were also chosen for control. Immunohistochemical stainings were performed to detect the expression of Bcl-2, Bax, Fas and Caspase-3 in those three groups. Endothelial cells were identified by expressing Factor related antigen. The average optical density and the rate of positive area of expression of Bcl-2, Bax, Fas and Caspase-3 were analyzed using image analysis Image HPIAS-
2000 Analysis System.
Results: The expression of Bcl-2 in proliferating hemangiomas was significantly higher than that in involuting hemangiomas and normal skin tissues(P<0.01). No significant difference was found between the expression of Bcl-2 in involuting hemangiomas and that in normal skin tissues (P>0.05). The expression of Bax, Fas and Caspase-3 in involuting hemangiomas was significantly higher than that in proliferating hemangiomas and normal skin tissues (P<0.01); the expression of Bax and Fas in proliferating hemangiomas is significantly higher than that in normal skin tissues (P<0.05). No significant difference was found between the expression of Caspase-3 in proliferating hema
方法：收集武汉大学人民医院病理科1996-2001年皮肤血管瘤存档蜡块49例，其中男性26例，女性23例，最小年龄2月，最大年龄68岁，平均年龄30.3岁。蜡块切片厚5μm，常规HE染色和免疫组织化学S-P法检测增殖细胞核抗原(proliferating cell nuclear antigen，PCNA)，按Mulliken分类标准并结合PCNA的表达进行分组，增生期血管瘤27例，退化期血管瘤22例，另取瘤组织周围正常皮肤组织5例作对照。运用免疫组织化学染色方法检测Bcl-2、Bax、Fas和Caspase-3在以上各组中的表达，并结合Ⅷ因子相关抗原的免疫组织化学染色证实表达Bcl-2、Bax、Fas和Caspase-3的
这些细胞是血管瘤组织中的内皮细胞。利用HPIAS一2000图像分析系统测定Bcl一2、
Bax、Fas和Caspase一3在以上三组中表达的平均光密度和平均阳性面积率。
结果:Bcl一2在增生期血管瘤内皮细胞的表达明显高于退化期血管瘤内皮细胞和正
常皮肤组织血管内皮细胞(尸<0.01);Bcl一2在退化期血管瘤内皮细胞的表达与正常皮
肤组织血管内皮细胞相比，差异无显著性(尸>0.05)。Bax、Fas和easpase一3在退化
期血管瘤内皮细胞的表达明显高于增生期血管瘤内皮细胞和正常皮肤组织血管内皮细
胞(尸<0.01);Bax和Fas在增生期血管瘤内皮细胞的表达高于正常皮肤组织血管内
皮细胞(尸<0.05);Caspase一3在增生期血管瘤内皮细胞的表达与正常皮肤组织血管内
皮细胞相比，差异无显著性(P>0.05)。
结论:Bcl一2、Bax、Fas和caspase一3参与了血管瘤的发生、发展和退化。Bcl一2通
过抑制内皮细胞凋亡而促进血管瘤的增生。Bax、Fas和Caspase一3通过诱导内皮细胞
凋亡而促进血管瘤由增生向退化的转变。
Objectives: Hemangioma, which is one of the commonest benign tumors, often occurs in infants and children. But the nomenclature and classification of hemangioma is confused in clinical diagnose before. Doctors always used 'hemangioma' to define various vascular diseases, including vascular malformations. Hemangioma is a kind of benign tumor formed by mass proliferative endothelial cells. The endothelial cell division of hemangioma increases aberrantly. And hemangioma has the tendency to involute spontaneously. Vascular malformation is a kind of structural abnormality during embryonal vasculogenesis. The endothelial cell division of vascular malformation is normal. And vascular malformation can not involute spontaneously. Based on the clinical observation and biological characters of the endothelial cells, the course of hemangioma can be divided into three phases: the proliferating, involuting and involuted phases. The mechanism of development and involution of hemangioma has not been clearly elucidated so fa
r. It has been pointed out that the development and involution of hemangioma are related to the proliferating status of hemangioma endothelium, the expression levels of various angiogenesis cytokines and angiogenesis inhibitors, and the imbalance of endothelial proliferation and apoptosis. The reasons for the spontaneous involution of hemangioma have raised more interest of researchers. The facts of much higher occurrence of apoptosis cells in hemangiomas than that in vascular malformations might provide some clues to explain the spontaneous involution of hemangiomas. Bcl-2, Bax, Fas and Caspase-3, which are hotspots in the researches on malignant tumorigenesis, have been reported to play important roles in apoptosis. As apoptosis is a crucial phenomenon in the course of hemangioma, we doubted whether these four proteins are also contributing to the developments and involution of hemangiomas. The study present here in showed the different expression patterns of Bcl-2, Bax, Fas and Caspase-3 in proliferating,
involuting hemangiomas and tissues from normal skin.
Methods: Specimens from 49 cases (26 males, and 23 females) of dermal hemangioma from the Department of Pathology in Renmin Hospital of Wuhan University from 1996 to 2001 were collected. The patients are from 2 months to 68 years old, with average age 30.3
iii
years old. The specimens were generally fixed by formalin, and embedded in paraffin. Sections of 5m thickness were made and attached to poly-L-lysine-coated glass slides. Hematoxylin and eosin (HE) staining were carried out routinely to identify the histological characters of hemangiomas. The phases of hemangiomas were identified by detecting the expression of proliferating cell nuclear antigen (PCNA) by immunohistochemistry. Based on Mulliken's standard, the hemangioma specimens were divided into two groups: proliferating hemangiomas (27 cases) and involuting hemangiomas (22 cases). Normal skin tissues around hemangiomas from 5 cases were also chosen for control. Immunohistochemical stainings were performed to detect the expression of Bcl-2, Bax, Fas and Caspase-3 in those three groups. Endothelial cells were identified by expressing Factor related antigen. The average optical density and the rate of positive area of expression of Bcl-2, Bax, Fas and Caspase-3 were analyzed using image analysis Image HPIAS-
2000 Analysis System.
Results: The expression of Bcl-2 in proliferating hemangiomas was significantly higher than that in involuting hemangiomas and normal skin tissues(P<0.01). No significant difference was found between the expression of Bcl-2 in involuting hemangiomas and that in normal skin tissues (P>0.05). The expression of Bax, Fas and Caspase-3 in involuting hemangiomas was significantly higher than that in proliferating hemangiomas and normal skin tissues (P<0.01); the expression of Bax and Fas in proliferating hemangiomas is significantly higher than that in normal skin tissues (P<0.05). No significant difference was found between the expression of Caspase-3 in proliferating hema
引文
1. 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): 2357-2364.
2. Chang J, Most D, Bresnick B, et al. Proliferative hemangiomas: analysis of cytokine gene expression and angiogenesis. Plast Reconstr Surg, 1999, 103(1): 1-9.
3．刘昱，张端莲，王瑞绵，等．雌激素受体、血管内皮生长因子和基质金属蛋白酶-2在血管瘤和血管畸形中的表达．解剖学杂志，2002，25(4)：354-359．
4．杨虹，周乾毅，张端莲，等．血管瘤组织中肥大细胞数量变化及意义．数理医药杂志，2001，14(3)：264-264．
5．洪莉，吴晔明，王燕，等．婴幼儿血管瘤自然消退过程中的细胞凋亡观察，中华小儿外科杂志，2002，23(3)：199-201．
6. Iwata J, Sonobe H, Furihata M, et al. High frequency of apoptosis in infantile capillary hemangioma. J Pathol, 1996, 179 (4): 403-408.
7. Razon MJ, Kraling BM, Mulliken JB, et al. Increased apoptosis coincides with onset of involution in infantile hemangioma. Microcirculation, 1998, 5 (2-3): 189-195.
8．程立新，梁杰，汤少明，等．细胞凋亡抑制基因bcl-2在血管瘤发病中作用的研究．中华整形烧伤外科杂志，1999，5(1)：35-36．
9. Hasan Q, Ruger BM, Tan ST, et al. Clusterin/apoJ expression during the development of hemangioma. Hum Pathol, 2000, 31 (6): 691-697.
10. Hyman KM, Seghezzi G, Pintucci G, et al. Transforming growth factor-betal induces apoptosis in vascular endothelial cells by activation of mitogen-activated protein kinase. Surgery, 2002, 132 (2): 173-179.
11. Sgonc R, Fuerhapter C, Boeck G, et al. Induction of apoptosis in human dermal microvascular endothelial cells and infantile hemangiomas by interferon-alpha. Int Arch Allergy Immunol, 1998, 117 (3): 209-214.
12．李向东，鲁开化，郭树忠，等．TNF-α在血管内皮细胞凋亡中的作用．第四军医大学学报，2001，22(12)：1078-1081．
13. O' Reilly MS, Tozawa Y, Deutsch U. Angioatatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. Cell, 1994, 79(4): 315-328
14. Mulliken JB, Glowacki J. Hemangiomas and vascular malformations in infants and children: a classification based on endothelial characteristics. Plast Reconstr Surg, 1982, 69(3): 412-422.
15. Mulliken JB, Young AE. Vascular birthmarks: hemangiomas and. malformations. Philadelphia: Saunders, 1988. 41-103.
16．金惠铭，卢建，殷莲华，主编．细胞分子病理生理学．郑州：郑州大学出版社，2002，369-370．
17. Tsujimoto Y, Finger LR, Yunis J, et al. Cloning of the chromosome breakpoint of neoplastic B cells with the t(14:18) chromosome translocation. Science, 1984, 226(4768): 1097-1099.
18. Vaux DL, Cory S, Adams JM. Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-Myc to immortalise pre-B cells. Nature 1988, 335(6189): 440-442
19. Hockenbery DM, Oltvai ZN, Yin XM, et al. Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell 1993 75(2): 241-251.
20. Lam M, Dubyak G, Chen L, et al. Evidence that Bcl-2 represses apoptosis by regulating endoplasmic reticulum-associated Ca~(2+)~fluxes. Proc Natl Acad Sci USA 1994, 91(14): 6569-6573.
21. Kluck RM, Bossy-Wetzel E, Green DR, et al. The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science, 1997, 275(5303): 1132-1136.
22. Oltvai ZN, Milliman CL, Korsmeyer SJ. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell dearth. Cell, 1993, 74(4): 609-619.
23. Zha H, Fisk HA, Yaffe MP, et al. Structure-function comparisons of the proapoptotic protein Bax in yeast and mammalian cells. Mol Cell Biol, 1996, 16(11): 6494-6508.
24．彭黎明，王曾礼，主编．细胞凋亡的基础与临床．北京：人民卫生出版社，2000：125-126．
25. Chao DT, Korsmeyer SJ. Bcl-2 Family: regulators of cell death. Annu Rev Immunol, 1998, 16: 395-419.
26. Turner HE, Nagy Z, Gatter KC, et al. Proliferation, bcl-2 expression and angiogenesis in pituitary adenoma: relationship to tumor behavior. Br J Cancer, 2000, 82(8): 1441-1445.
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38. Perera L, Waldmann TA. Activation of human monocytes induces differential resistance to apoptosis with rapid down regulation of caspase-8/FLICE. Proc Natl Acad Sci USA, 1998, 95(24): 14308-14313.
2. Chang J, Most D, Bresnick B, et al. Proliferative hemangiomas: analysis of cytokine gene expression and angiogenesis. Plast Reconstr Surg, 1999, 103(1): 1-9.
3．刘昱，张端莲，王瑞绵，等．雌激素受体、血管内皮生长因子和基质金属蛋白酶-2在血管瘤和血管畸形中的表达．解剖学杂志，2002，25(4)：354-359．
4．杨虹，周乾毅，张端莲，等．血管瘤组织中肥大细胞数量变化及意义．数理医药杂志，2001，14(3)：264-264．
5．洪莉，吴晔明，王燕，等．婴幼儿血管瘤自然消退过程中的细胞凋亡观察，中华小儿外科杂志，2002，23(3)：199-201．
6. Iwata J, Sonobe H, Furihata M, et al. High frequency of apoptosis in infantile capillary hemangioma. J Pathol, 1996, 179 (4): 403-408.
7. Razon MJ, Kraling BM, Mulliken JB, et al. Increased apoptosis coincides with onset of involution in infantile hemangioma. Microcirculation, 1998, 5 (2-3): 189-195.
8．程立新，梁杰，汤少明，等．细胞凋亡抑制基因bcl-2在血管瘤发病中作用的研究．中华整形烧伤外科杂志，1999，5(1)：35-36．
9. Hasan Q, Ruger BM, Tan ST, et al. Clusterin/apoJ expression during the development of hemangioma. Hum Pathol, 2000, 31 (6): 691-697.
10. Hyman KM, Seghezzi G, Pintucci G, et al. Transforming growth factor-betal induces apoptosis in vascular endothelial cells by activation of mitogen-activated protein kinase. Surgery, 2002, 132 (2): 173-179.
11. Sgonc R, Fuerhapter C, Boeck G, et al. Induction of apoptosis in human dermal microvascular endothelial cells and infantile hemangiomas by interferon-alpha. Int Arch Allergy Immunol, 1998, 117 (3): 209-214.
12．李向东，鲁开化，郭树忠，等．TNF-α在血管内皮细胞凋亡中的作用．第四军医大学学报，2001，22(12)：1078-1081．
13. O' Reilly MS, Tozawa Y, Deutsch U. Angioatatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. Cell, 1994, 79(4): 315-328
14. Mulliken JB, Glowacki J. Hemangiomas and vascular malformations in infants and children: a classification based on endothelial characteristics. Plast Reconstr Surg, 1982, 69(3): 412-422.
15. Mulliken JB, Young AE. Vascular birthmarks: hemangiomas and. malformations. Philadelphia: Saunders, 1988. 41-103.
16．金惠铭，卢建，殷莲华，主编．细胞分子病理生理学．郑州：郑州大学出版社，2002，369-370．
17. Tsujimoto Y, Finger LR, Yunis J, et al. Cloning of the chromosome breakpoint of neoplastic B cells with the t(14:18) chromosome translocation. Science, 1984, 226(4768): 1097-1099.
18. Vaux DL, Cory S, Adams JM. Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-Myc to immortalise pre-B cells. Nature 1988, 335(6189): 440-442
19. Hockenbery DM, Oltvai ZN, Yin XM, et al. Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell 1993 75(2): 241-251.
20. Lam M, Dubyak G, Chen L, et al. Evidence that Bcl-2 represses apoptosis by regulating endoplasmic reticulum-associated Ca~(2+)~fluxes. Proc Natl Acad Sci USA 1994, 91(14): 6569-6573.
21. Kluck RM, Bossy-Wetzel E, Green DR, et al. The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science, 1997, 275(5303): 1132-1136.
22. Oltvai ZN, Milliman CL, Korsmeyer SJ. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell dearth. Cell, 1993, 74(4): 609-619.
23. Zha H, Fisk HA, Yaffe MP, et al. Structure-function comparisons of the proapoptotic protein Bax in yeast and mammalian cells. Mol Cell Biol, 1996, 16(11): 6494-6508.
24．彭黎明，王曾礼，主编．细胞凋亡的基础与临床．北京：人民卫生出版社，2000：125-126．
25. Chao DT, Korsmeyer SJ. Bcl-2 Family: regulators of cell death. Annu Rev Immunol, 1998, 16: 395-419.
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