平阳霉素乳剂对增生期毛细血管瘤细胞凋亡的影响
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摘要
血管瘤是婴幼儿时期最常见的一种良性软组织肿瘤，国内习惯上按照Mulliken的分类法将其分为真性血管瘤（hemangioma）和血管畸形（vascular malformation）。真性血管瘤包括毛细血管瘤和混合型血管瘤，而后者则包括海绵状血管瘤和蔓状血管瘤。我们所讨论的则为最常见的毛细血管瘤（约占50%~60%），其自然病程可分为增生期和消退期，由于其发病率高，生长迅速，发生部位较广泛等特点，可对患者的生理功能甚至生命造成严重的威胁，因而如何有效的治疗毛细血管瘤是十分迫切和必要的。传统的手术切除、激光、冷冻、同位素等治疗方法都为侵袭性的，疗效均不十分满意，且对生长于颜面部的瘤体尤为不宜。而平阳霉素（pingyangmycin , PYM）是一种国产的抗肿瘤抗生素,作为化疗药物，无免疫抑制作用，对骨髓造血功能也较少抑制，较适用于血管瘤的治疗,是目前一种较为流行的治疗血管瘤的药物。大量文献证实PYM可促进恶性肿瘤组织细胞发生凋亡而达到治疗目的，而毛细血管瘤具有幼稚胚胎血管内皮的特征，是胚胎废用性组织的异常增生，国内外研究表明在血管瘤中可检出细胞凋亡的表达，尤其在消退期则更为明显，因而其本身就存在自行凋亡而消退的趋势。细胞凋亡是生物体内的一个主动的程序化细胞死亡过
程，是机体清除生理上不需要细胞的重要方式，参与机体废用组织的生理性萎缩和消散。所以我们拟将PYM制成一定浓度的乳剂以减少传统的PYM 瘤体内注射治疗所引起的皮肤纤维化甚至坏死等副作用，继而研究PYM乳剂是否能有效地促进增生期毛细血管瘤组织发生凋亡而加速其进入消退期继而消亡，从而达到安全有效的治疗目的。根据检索大量文献，目前国内外未发现有此同类研究。
目的：探讨平阳霉素乳剂对增生期毛细血管瘤组织细胞凋亡的影响，从分子水平进一步探讨平阳霉素治疗血管瘤的机制，从而为血管瘤的治疗寻找有效的途径。
方法：试验对象选取均为6月以内的婴儿体表毛细血管瘤30例，将其随机分为A、B两组，每组15例。A组为对照组，B组为平阳霉素用药组。A、B两组受试患儿均行胸透和血常规检查示结果正常后，将平阳霉素制成一定浓度的乳剂均匀涂抹于B组每个试验对象的瘤体表面。表面再敷以塑料贴膜以防止药物蒸发干燥，每日3次，平均涂药7天左右。A组则不用任何药物治疗。7天时，A、B两组受试对象均以手术方式将其瘤体完整切除，切除瘤体以10%中性甲醛固定，制成石蜡切片观察细胞微观结构变化，继而进一步行分子生物学试验（TUNEL试验）检测A、B两组细胞凋亡指数（apoptotic index，A I）。另分别取A、B两组各2例标本以4%戊二醛固定送电镜观察细胞超微结构变化。
结果：1、大体观察：A组受试标本大体无明显改变；B组中约2/3受试标本在涂用平阳霉素乳剂治疗后，瘤
体颜色较前变暗，表面皮肤发生轻微皱缩，而其余1/3则无明显变化。2、光镜病理观察：可见A组血管瘤内皮细胞增生活跃，可形成内皮细胞团块样结构，血管腔可被阻塞，血管呈丛状或小叶状分布。B组血管瘤内皮细胞成分减少，可见散在呈岛状分布的脂肪和纤维组织，内皮细胞成束状排列，可见血管腔。3、电镜观察：A组血管瘤内细胞增生活跃，可见内皮细胞聚集成团，细胞之间排列紧密，很少间隙或其他成分，内皮细胞外形大小正常，胞浆均匀，染色质均匀分布，可见较多地线粒体、内质网和高尔基复合体等各种细胞器，其外形结构正常。视野中少见凋亡细胞，而肥大细胞则丰富多见。其功能旺盛，富含分泌颗粒。甚至可见到肥大细胞群集于毛细血管周围的现象。B组中则多见凋亡的内皮细胞，其外形缩小，和周围细胞失去连接，可见染色质边集形成凋亡起始标志——指状突起，染色质进一步边集于核膜下，胞浆致密化，可见新月体的形成。甚至出现核固缩，核膜破裂，形成凋亡小体等细胞凋亡现象。线粒体等细胞器互相靠近，部分也发生致密固缩。视野中肥大细胞较少见。4、分子生物学试验结果显示：A组标本TUNEL试验染色后可见少量散在分布的凋亡细胞，核呈棕黄色，而周围则是核呈蓝色的正常细胞；B组标本染色后可见较大量的凋亡细胞，表现为细胞皱缩，体积缩小，甚至有核固缩现象。部分凋亡细胞位于血管腔表面，且趋于形成管腔结构的细胞发生凋亡者居多。A组血管瘤内细胞的凋亡指数为9.693±4.948。B组凋亡指数为39.373±15.927。两者比较，B组血管瘤内细胞凋
亡率较A组明显增高，有非常显著性差异（P<0.01）。
结论：毛细血管瘤存在自行消亡的趋势，平阳霉素乳剂作为治疗血管瘤的一种有效方法，可促进其细胞发生凋亡，从而加速其消退，缩短其病程，达到治疗目的。其促进血管瘤细胞（主要为内皮细胞）发生凋亡的机制可能与其选择性的激活凋亡信号传导通路有关。
Infantile capillary hemangioma (ICH) is one of the most common benign inalacoid tumors of infancy. they could bring about all kings of damage because they occur in every place of one body with a high morbidity and rapidly proliferation. Therefore , it is urgent to cure them timely and effectively .Pingyangmycin(PYM) is a kind of popular drug which is used for hemangiomas' treatment. But the traditional way of PYM being injected into tumor has more side effects, so PYM was made into emulsion and smeared the surfaces of the tumors evenly in order to reduce the extra damage. Many documents suggest that PYM can induce apoptosis in malignant tumor in order to cure them .Furthermore , the tendency that capillary hemangiomas gradually wither away with themselves in their course of disease is evidence . So that , it is necessary to study whether PYM emulsion effectively promote apoptosis in hemangiomas in order that the tumors could die more rapidly.
Objective : To insight whether PYM emulsion induce apoptosis more rapidly in proliferating capillary
hemangiomas and further inquiry the mechanism at molecular level so as to find the optimal method for hemangiomas' treatment.
Method: The specimens were all the epizoic tumors which were obtained from the children whose ages were all lower than 6 months . They were divided into 2 groups , A and B, every group counts 15. Group A was used as control , whereas group B were experimental group. PYM was made into emulsion and smeared the surfaces of the tumors. This course lasted about 7 days, 3 times every day . The specimens were resected by operation on day 7,then made into pathological slices and electron microscope slices . Furthermore , the apoptotic cells in group A and B were tested by molecular biology method (TUNEL test ).
Results: 1. About 2/3 of group B became deep red and their surfaces began to shrink slightly after having been treated by PYM emulsion . Those of group A kept the same as before . 2. Light micrographs showed that the endothelial cells of group A proliferated actively and tended to gather together like a ball, the capillary lumens were closed up and the little vascular scattered fasciculatly or foliolarly . However , the endothelial cells of group B became less and ranked bunchily and the lumens was easily seen . There were adipose tissue and fibrous tissue between the cells .3. The results of electron micrographs : the cells of group A proliferated actively and closed together whose
appearances were normal and chromatin distributed evenly . There were abundant organelles such as mitochondria , endoplasmicreticulum and golgi complex in the cells . The apoptotic cells were few while the mast cells was easily seen that gathered beside the capillary lumens . On the contrary ,There were much apoptotic phenomena in group B: The external forms of the cells became shrinkage , the chromatin assembled near the nuclear membrane and formed crescent body, the cell-cell junctions disappeared and cytoplasm became condense , all kinds of organcelles condensed and closed each other . Furthermore , pycnosis and caryoclasis were seen in the test . In addition ,we also found the apoptotic-initating signal―chromatin phalangeal process. The mast cells became less than that of group A. 4. The results of TUNEL test were agree with those of electron micrographs . There was a small amount of yellow -brown apoptotic cells scattering among the blue normal cells in group A.The cells of group B showed the higher apoptotic rate. Part of apoptotic cells lied in the surfaces of the lumens .The more close to the lumens , the more high rate of apoptosis. The results of statistics showed that the apoptotic index (AI) of group A was 9.693±4.948 and that of group B was 39.373±15.927, there was extraodinary significant diffrence between A and B (P<0.01).
Condusion : ICH present a tendency of spantaneous apoptosis . As a way of curing hemangiomas ,PYM
emulsion can effectively accelerate apoptosis majorly in their endothelial cells
程，是机体清除生理上不需要细胞的重要方式，参与机体废用组织的生理性萎缩和消散。所以我们拟将PYM制成一定浓度的乳剂以减少传统的PYM 瘤体内注射治疗所引起的皮肤纤维化甚至坏死等副作用，继而研究PYM乳剂是否能有效地促进增生期毛细血管瘤组织发生凋亡而加速其进入消退期继而消亡，从而达到安全有效的治疗目的。根据检索大量文献，目前国内外未发现有此同类研究。
目的：探讨平阳霉素乳剂对增生期毛细血管瘤组织细胞凋亡的影响，从分子水平进一步探讨平阳霉素治疗血管瘤的机制，从而为血管瘤的治疗寻找有效的途径。
方法：试验对象选取均为6月以内的婴儿体表毛细血管瘤30例，将其随机分为A、B两组，每组15例。A组为对照组，B组为平阳霉素用药组。A、B两组受试患儿均行胸透和血常规检查示结果正常后，将平阳霉素制成一定浓度的乳剂均匀涂抹于B组每个试验对象的瘤体表面。表面再敷以塑料贴膜以防止药物蒸发干燥，每日3次，平均涂药7天左右。A组则不用任何药物治疗。7天时，A、B两组受试对象均以手术方式将其瘤体完整切除，切除瘤体以10%中性甲醛固定，制成石蜡切片观察细胞微观结构变化，继而进一步行分子生物学试验（TUNEL试验）检测A、B两组细胞凋亡指数（apoptotic index，A I）。另分别取A、B两组各2例标本以4%戊二醛固定送电镜观察细胞超微结构变化。
结果：1、大体观察：A组受试标本大体无明显改变；B组中约2/3受试标本在涂用平阳霉素乳剂治疗后，瘤
体颜色较前变暗，表面皮肤发生轻微皱缩，而其余1/3则无明显变化。2、光镜病理观察：可见A组血管瘤内皮细胞增生活跃，可形成内皮细胞团块样结构，血管腔可被阻塞，血管呈丛状或小叶状分布。B组血管瘤内皮细胞成分减少，可见散在呈岛状分布的脂肪和纤维组织，内皮细胞成束状排列，可见血管腔。3、电镜观察：A组血管瘤内细胞增生活跃，可见内皮细胞聚集成团，细胞之间排列紧密，很少间隙或其他成分，内皮细胞外形大小正常，胞浆均匀，染色质均匀分布，可见较多地线粒体、内质网和高尔基复合体等各种细胞器，其外形结构正常。视野中少见凋亡细胞，而肥大细胞则丰富多见。其功能旺盛，富含分泌颗粒。甚至可见到肥大细胞群集于毛细血管周围的现象。B组中则多见凋亡的内皮细胞，其外形缩小，和周围细胞失去连接，可见染色质边集形成凋亡起始标志——指状突起，染色质进一步边集于核膜下，胞浆致密化，可见新月体的形成。甚至出现核固缩，核膜破裂，形成凋亡小体等细胞凋亡现象。线粒体等细胞器互相靠近，部分也发生致密固缩。视野中肥大细胞较少见。4、分子生物学试验结果显示：A组标本TUNEL试验染色后可见少量散在分布的凋亡细胞，核呈棕黄色，而周围则是核呈蓝色的正常细胞；B组标本染色后可见较大量的凋亡细胞，表现为细胞皱缩，体积缩小，甚至有核固缩现象。部分凋亡细胞位于血管腔表面，且趋于形成管腔结构的细胞发生凋亡者居多。A组血管瘤内细胞的凋亡指数为9.693±4.948。B组凋亡指数为39.373±15.927。两者比较，B组血管瘤内细胞凋
亡率较A组明显增高，有非常显著性差异（P<0.01）。
结论：毛细血管瘤存在自行消亡的趋势，平阳霉素乳剂作为治疗血管瘤的一种有效方法，可促进其细胞发生凋亡，从而加速其消退，缩短其病程，达到治疗目的。其促进血管瘤细胞（主要为内皮细胞）发生凋亡的机制可能与其选择性的激活凋亡信号传导通路有关。
Infantile capillary hemangioma (ICH) is one of the most common benign inalacoid tumors of infancy. they could bring about all kings of damage because they occur in every place of one body with a high morbidity and rapidly proliferation. Therefore , it is urgent to cure them timely and effectively .Pingyangmycin(PYM) is a kind of popular drug which is used for hemangiomas' treatment. But the traditional way of PYM being injected into tumor has more side effects, so PYM was made into emulsion and smeared the surfaces of the tumors evenly in order to reduce the extra damage. Many documents suggest that PYM can induce apoptosis in malignant tumor in order to cure them .Furthermore , the tendency that capillary hemangiomas gradually wither away with themselves in their course of disease is evidence . So that , it is necessary to study whether PYM emulsion effectively promote apoptosis in hemangiomas in order that the tumors could die more rapidly.
Objective : To insight whether PYM emulsion induce apoptosis more rapidly in proliferating capillary
hemangiomas and further inquiry the mechanism at molecular level so as to find the optimal method for hemangiomas' treatment.
Method: The specimens were all the epizoic tumors which were obtained from the children whose ages were all lower than 6 months . They were divided into 2 groups , A and B, every group counts 15. Group A was used as control , whereas group B were experimental group. PYM was made into emulsion and smeared the surfaces of the tumors. This course lasted about 7 days, 3 times every day . The specimens were resected by operation on day 7,then made into pathological slices and electron microscope slices . Furthermore , the apoptotic cells in group A and B were tested by molecular biology method (TUNEL test ).
Results: 1. About 2/3 of group B became deep red and their surfaces began to shrink slightly after having been treated by PYM emulsion . Those of group A kept the same as before . 2. Light micrographs showed that the endothelial cells of group A proliferated actively and tended to gather together like a ball, the capillary lumens were closed up and the little vascular scattered fasciculatly or foliolarly . However , the endothelial cells of group B became less and ranked bunchily and the lumens was easily seen . There were adipose tissue and fibrous tissue between the cells .3. The results of electron micrographs : the cells of group A proliferated actively and closed together whose
appearances were normal and chromatin distributed evenly . There were abundant organelles such as mitochondria , endoplasmicreticulum and golgi complex in the cells . The apoptotic cells were few while the mast cells was easily seen that gathered beside the capillary lumens . On the contrary ,There were much apoptotic phenomena in group B: The external forms of the cells became shrinkage , the chromatin assembled near the nuclear membrane and formed crescent body, the cell-cell junctions disappeared and cytoplasm became condense , all kinds of organcelles condensed and closed each other . Furthermore , pycnosis and caryoclasis were seen in the test . In addition ,we also found the apoptotic-initating signal―chromatin phalangeal process. The mast cells became less than that of group A. 4. The results of TUNEL test were agree with those of electron micrographs . There was a small amount of yellow -brown apoptotic cells scattering among the blue normal cells in group A.The cells of group B showed the higher apoptotic rate. Part of apoptotic cells lied in the surfaces of the lumens .The more close to the lumens , the more high rate of apoptosis. The results of statistics showed that the apoptotic index (AI) of group A was 9.693±4.948 and that of group B was 39.373±15.927, there was extraodinary significant diffrence between A and B (P<0.01).
Condusion : ICH present a tendency of spantaneous apoptosis . As a way of curing hemangiomas ,PYM
emulsion can effectively accelerate apoptosis majorly in their endothelial cells
引文
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6 Mang Collins FIM, Julie Glowacki PHD, John B,etal . Congential vascular lesions: clinical application of a new classification. Journal of Pediatr Surg, 1983, vol18(6):894~900
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2 彭心昭,沈忠英,黎妙娟等. 平阳霉素诱导人食管癌细胞EC8712凋亡的研究. 《癌症》,1999,16(6):657~660
3 洪莉 , 吴晔明 , 王燕 等 . 婴幼儿血管瘤自然消退过程中细胞凋亡观察. 中华小儿外科杂志, 2002,6,23(3):199~201
4 John B, Mulliken MD, Julie Glowacki phD. Hemangiomas and vascular malformation in infants and children: A classification based on endothelial characteristics. Plast and Reconstr Surg, 1982,vol69(3):412~422
5 John B, Mulliken MD, Bruce R , etal In vitro characteristics of endothelium from hemangiomas and vascular malformations. Surgery, 1982,92(2):348~353
6 Mang Collins FIM, Julie Glowacki PHD, John B,etal . Congential vascular lesions: clinical application of a new classification. Journal of Pediatr Surg, 1983, vol18(6):894~900
7 高解春,陆慧君,金百祥. 婴儿血管瘤自然消退的临床过程. 中华小儿外科杂志, 1992,13(3):139~141
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