【作者】 董学易；

【导师】 孙保存； 赵秀兰； 孙涛；

【作者基本信息】 天津医科大学 ， 病理学与病理生理学， 2010， 硕士

【摘要】 研究目的：1)对人体石蜡组织标本的回顾性研究,研究人体的黑色素瘤组织中是否存在VM和LPPCN,并分析VM和LPPCN与黑色素瘤患者临床病理参数及预后之间的关系,在此基础上阐述LPPCN和VM在黑色素瘤患者中的临床病理意义；2)初步研究凋亡相关蛋白在黑色素瘤组织中的表达并分析它们在LPPCN形成中所起的作用,在此基础上进一步探讨LPPCN形成的相关分子机制；3)研究肿瘤侵袭与转移相关蛋白Twist1、MMP-2、MMP-9在黑色素瘤组织中的表达并分析它们在LPPCN和VM形成过程中所起的作用。研究方法：1)收集天津医科大学附属肿瘤医院1996年1月5日-2007年11月26日期间符合实验要求的黑色素瘤标本70例,收集患者的发病年龄、性别、肿瘤大小、发病部位、复发与转移等临床资料,总结分析黑色素瘤患者的临床病理特征；2)利用HE染色及CD31／PAS双重染色的方法观察70例黑色素瘤组织中是否存在VM和LPPCN,并在显微镜下计数各自的分布情况,用散点图和相关检验法分析LPPCN计数、VM计数、MVD计数之间是否存在相关。并在此基础上进一步分析VM和LPPCN的存在与黑色素瘤患者的临床病理参数及预后之间的关系,LPPCN与VM、内皮依赖性血管之间的关联性；3)用免疫组化染色的方法检测凋亡相关蛋白在SPPCN细胞、LPPCN细胞及它们周围肿瘤细胞中的表达水平,侵袭与转移相关蛋白MMP-2、MMP-9、Twist1在LPPCN (-)组和LPPCN (+)组、VM(-)组和VM(+)组中的表达水平,用染色指数的方法评价这些蛋白在组间的表达有无差异,并进一步分析这些蛋白的表达在LPPCN和VM形成过程中所起的作用。结果：1黑色素瘤组织中存在VM和LPPCN1.1黑色素瘤组织的形态学观察结果对黑色素瘤组织进行HE染色和CD31/PAS双重染色结果显示：70例黑色素瘤有33例中存在VM,38例中存在LPPCN,22例中同时存在SPPCN和LPPCN的现象。1.2 VM和LPPCN与黑色素瘤患者临床病理参数及预后的关系VM和LPPCN的发生与患者的年龄、性别、肿瘤生长部位、有无黑色素颗粒、复发和远处转移无关(P>0.05),与患者的肿瘤体积有关(P<0.05)。当肿瘤体积大于20.41cm3的时候比较容易发生VM和LPPCN(P<0.05)。Kaplan-Meier生存曲线分析结果显示VM(+)组较VM(-)组、LPPCN(+)组较LPPCN(-)组、VM+LPPCN(+)组较VM+LPPCN(-)组患者的生存时间短,Log-rank检验法比较各组间的生存曲线的差异有统计学意义(P<0.05)。1.3黑色素瘤组织中VM计数、LPPCN计数与MVD计数之间的关系VM(+)组MVD计数的平均值高于VM(-)组MVD计数的平均值(P<0.05)。LPPCN(-)组与LPPCN(+)组之间的MVD计数平均值差异比较无统计学意义(P>0.05)。VM+LPPCN(+)组MVD计数的平均值高于VM+LPPCN(-)组MVD计数的平均值(P<0.05)。2各参数之间相关分析结果VM计数与LPPCN计数、LPPCN细胞计数之间存在正相关(r=0.469,r=0.470；P=0.024,P=-0.024)。LPPCN计数与LPPCN细胞计数之间存在正相关(r=0.406,P=0.011),肿瘤体积与MVD计数之间存在正相关(r=0.261,P=0.029)。3凋亡相关蛋白的免疫组化染色结果3.1线粒体凋亡通路相关蛋白在黑色素瘤组织中的表达3.1.1 Bcl-2和Bax蛋白在黑色素瘤组织中的表达水平Bcl-2蛋白在LPPCN(+)组的表达高于LPPCN(-)组的表达(P<0.05)。在LPPCN形成的过程中,早期的SPPCN细胞中Bcl-2蛋白的表达高于晚期LPPCN细胞中Bcl-2蛋白的表达(P<0.05)。早期的SPPCN细胞中Bax蛋白的表达低于晚期LPPCN细胞中Bax蛋白的表达(P<0.05)。早期的SPPCN周围肿瘤细胞中Bcl-2蛋白的表达低于晚期LPPCN周围肿瘤细胞中Bcl-2蛋白的表达(P<0.05)。早期的SPPCN周围肿瘤细胞中Bax蛋白的表达高于晚期LPPCN周围肿瘤细胞中Bax蛋白的表达(P<0.05)。在早期阶段,SPPCN细胞中Bcl-2和Bax蛋白的表达与SPPCN周围肿瘤细胞中Bcl-2和Bax蛋白的表达差异比较无统计学意义(P>0.05)。在晚期阶段,LPPCN细胞中Bcl-2蛋白的表达低于LPPCN周围肿瘤细胞中Bcl-2蛋白的表达(P<0.05)。LPPCN细胞中Bax蛋白的表达高于LPPCN周围肿瘤细胞中Bax蛋白的表达(P<0.05)。3.1.2 Caspase9和Caspase3蛋白在黑色素瘤组织中的表达线粒体凋亡途径相关蛋白Caspase9和Caspase3在LPPCN(-)组与LPPCN(+)组之间表达差异比较无统计学意义(P>0.05)。Caspase9和Caspase3蛋白在LPPCN细胞中的表达高于LPPCN周围肿瘤细胞中的表达(P<0.05)。3.2肿瘤坏死因子家族相关蛋白在黑色素瘤组织中的表达膜受体凋亡途径相关蛋白TNF、TRAIL、Fas、FasL、Caspase8在LPPCN(-)组与LPPCN (+)组之间表达差异比较无统计学意义(P>0.05)。TNF、TRAIL、Fas、FasL、Caspase8蛋白在LPPCN细胞中的表达与LPPCN周围肿瘤细胞中的表达差异比较无统计学意义(P>0.05)。4侵袭与转移相关蛋白Twist1、MMP-2、MMP-9的免疫组化染色结果Twist1蛋白在LPPCN(+)组中的表达高于LPPCN(-)组中的表达(P<0.05),在VM(+)组中的表达高于VM(-)组中的表达(P<0.05),在LPPCN细胞中的表达高于LPPCN周围肿瘤细胞的表达(P<0.05)。MMP-2蛋白在LPPCN(+)组中的表达高于LPPCN (-)组中的表达(P<0.05),在VM(+)组中的表达高于VM(-)组中的表达(P<0.05)。MMP-9蛋白在VM(+)组中的表达高于VM(-)组中的表达(P<0.05)。结论1黑色素瘤组织中存在VM和LPPCN,存在VM或／和LPPCN的黑色素瘤患者的临床预后差。VM和LPPCN的发生与患者的年龄、性别、发生部位、有无黑色素颗粒、复发与远处转移无关。当肿瘤的体积>20.41cm3(直径为3cm左右)时容易发生VM和LPPCN.2 LPPCN是肿瘤细胞为适应环境产生的一种介于凋亡和坏死之间过渡的细胞死亡形式。在缺氧的环境下,部分瘤细胞在线粒体凋亡通路为主的调控下先形成SPPCN,随着缺氧程度的加剧,SPPCN发展为LPPCN。3黑色素瘤细胞表达的MMP-9、MMP-2、Twistl蛋白都参与了VM形成过程中的细胞形变和基质重塑。肿瘤细胞分泌的PAS阳性物质参与了VM形成中的基质重塑。更多还原

【Abstract】 Objective:1) To identify if VM and LPPCN existing in human melanoma through a retrospective study with paraffin tissue samples. To explore the correlation between LPPCN, VM and clinicopathologic factors, clinical prognosis in melanoma’patients. Based on these, to investigate the clinical and pathological significance of VM and LPPCN in melanoma’patients.2) To disclose the role of apoptosis-associated proteins and analyze relevant molecular mechanism in LPPCN formation.3) To investigate the expression of Twist1, MMP-2, MMP-9 in melanoma and analyze their role in LPPCN and VM formation.Methods:1) 70 melanoma cases undergoing surgery in Tumor Hospital affiliated of Tianjin Medical University from January 1996 to November 2007 were collected in this study. The information of samples including age, gender, tumor size, location, recurrence and metastasis were collected and analyzed the clinicopathologic character of patients.2) CD31/PAS double staining and HE staining was performed on the slides to confirm the existence of VM and LPPCN. Besides, to count the VM, LPPCN, MVD and analyze the relation between them in melanoma. Based on these, to explore the correlation between LPPCN,VM and clinicopathologic parameter、clinical prognosis in melanoma’patients and investigate the association of LPPCN and VM, endothelium-dependent vessels.3) Immunohistochemistry method was used to detect the expression of apoptosis-associated proteins in scatter patterned programmed cell necrosis cells, Linearly patterned programmed cell necrosis cells and their circum-tumors cells and the expression of invasive-associated proteins in LPPCN-positive group and LPPCN-negative group, VM-positive group and VM-negative group. Staining index was used to evaluate the difference of these proteins expressed and analyzing their role in VM and LPPCN formation.Results:1 VM and LPPCN exists in melanoma1.1 The morphological observed results in melanomaCD31/PAS double staining and HE staining shows 33 cases existed VM and 38 cases existed LPPCN in 70 cases melanoma. Besides,22 cases existed SPPCN and LPPCN.1.2 The relation between LPPCN、VM and clinicopathologic data in melanomaThe occurrence of VM and LPPCN was not correlated with age, gender, location, melanin pigment recurrence and metastasis (P>0.05).The melanoma was prone to generate VM and LPPCN when its volume exceed 20.41cm3(P<0.05) Kaplan-Meier survive plot shows the survive time of VM-positive group was shorter than VM-negative group, the survive time of LPPCN-positive group was shorter than LPPCN-negative group, the survive time of VM and LPPCN-positive group was shorter than VM and LPPCN-negative group (P<0.05)。1.3 The relation between LPPCN、VM and MVD counting in melanomaThe average MVD counting in VM-positive group was higher than that VM-negative group (P<0.05). The average MVD counting in VM and LPPCN-positive group was higher than that VM and LPPCN-negative group (P<0.05)2 The correlation results of every parameterThere is a highly positive correlation between the number of VM and the number of LPPCN, the number of LPPCN cells (r=0.469, r=0.470;P=0.024, P=0.024). There is a highly positive correlation between the number of LPPCN and the number of LPPCN cells (r=0.406, P=0.011).There is a highly positive correlation between the size of tumor and the number of endothelium-dependent vessels(r=0.261, P=0.029).3 The immunohistochemisty staining results of apoptosis-associated proteins3.1 The expression of mitochondrial-mediating apoptosis relative proteins3.1.1 The expression of Bcl-2 and Bax in melanomaThe expression of Bcl-2 in LPPCN-positive group was higher than LPPCN--negative group (P<0.05).The expression of Bcl-2 in SPPCN cells was higher than LPPCN cells (P<0.05). The expression of Bax in SPPCN cells was lower than LPPCN cells (P<0.05). The expression of Bcl-2 in SPPCN circum-tumors cells was lower than LPPCN circum-tumors cells (P<0.05). The expression of Bax in SPPCN circum-tumors cells was higher than LPPCN circum-tumors cells (P<0.05). The expression of Bcl-2 in LPPCN cells was lower than LPPCN circum-tumors cells (P<0.05). The expression of Bax in LPPCN cells was higher than LPPCN circum-tumors cells (P<0.05)。3.1.2 The expression of Caspase9 and Caspase3 in melanomaThe expression of Caspase9 and Caspase3 in LPPCN cells was higher than LPPCN circum-tumors cells(P<0.05). The expression of Caspase9 and Caspase3 in LPPCN-negative group and LPPCN-positive group had not statistical significance (P>0.05).3.2 The expression of tumor necrosis factor family-mediating apoptosis relative-proteins in melanomaThe expression of TNF, TRAIL, Fas, FasL,Caspase8 in LPPCN-negative group and LPPCN-positive group had not statistical significance(.P>0.05). The expression of TNF, TRAIL, Fas, FasL, Caspase8 in LPPCN cells and LPPCN circum-tumors cells had not statistical significance (P>0.05)4 The expression of Twist1,MMP2,MMP-9 in melanomaThe expression of Twistl in LPPCN-positive group was higher than LPPCN-negative group (P<0.05).The expression of Twist1 in VM-positive group was higher than VM-negative group (P<0.05). The expression of Twist1 in LPPCN cells was higher than LPPCN circum-tumors cells (P<0.05).The expression of MMP-2 and MMP-9 in VM-positive group was higher than VM-negative group (P<0.05). The expression of MMP-2 in LPPCN-positive group was higher than LPPCN-negative group (P<0.05)Conclusion:1 VM and LPPCN existed in melanoma and the patients with VM or/and LPPCN has worse clinical prognosis than those without VM and LPPCN. The occurrence of VM and LPPCN was not correlated with age, gender, location, melanin pigment, recurrence and metastasis. The melanoma was prone to generate VM and LPPCN when its volume exceeds 20.41cm3 (diameters about 3 mm).2 LPPCN is a transitive cells’dead way between apoptosis and necrosis. Under the genes control of mitochondrial-mediating apoptosis, some melanoma cells underwent SPPCN by hypoxic condition. SPPCN will develop LPPCN when tumor tissue lacks oxygen severely.3 The proteins of MMP-9,MMP-2,Twist1 expressed by melanoma play a important role in VM formation. The melanoma cells could secrete PAS matter and it participates stromatic remodeling of VM.更多还原