人体黑色素瘤中LPPCN与VM的关系及其调控机制的初步研究

发布时间：2018-02-17 04:24

本文关键词： 黑色素瘤线形程序性坏死血管生成拟态血管内皮生长因子受体　出处：《天津医科大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的研究人体黑色素瘤组织中是否存在线形程序性坏死(linearly patterned programmed cell necrosis, LPPCN)及血管生成拟态(vasculogenic mimicry, VM)及其分布情况,分析它们的临床病理意义。研究70例人体黑色素瘤组织中LPPCN及VM阳性病例中的LPPCN、VM周围肿瘤细胞血.管内皮生长因子(VEGF)及其受体VEGFR1 (Flt-1)和VEGFR2 (Flk-1)的表达情况并研究VEGF与侵袭转移相关蛋白的相互作用,探讨它们在肿瘤血管生成中的作用以及与临床预后的关系。方法 1、自1996-2007年天津医科大学附属肿瘤医院存档石蜡标本中挑选出符合实验要求(经手术切除、病理诊断明确且随访资料完整)的恶性黑色素瘤标本共70例,收集患者的性别、年龄、发病部位、肿瘤大小、手术日期、复发与转移和随访等临床资料。分析黑色素瘤患者的临床病理特征及其与预后的关系； 2、利用HE染色观察70例黑色素瘤组织中是否存在LPPCN及利用CD31/PAS双重染色的方法来观察这70例黑色素瘤组织中是否存在VM,并在显微镜下计数其各自的分布情况。运用相关检验法分析LPPCN、VM与MVD之间有无相关以探讨LPPCN与VM、内皮依赖性血管之间的关联性,并在此基础上进一步分析LPPCN、VM的存在与黑色素瘤患者的临床病理参数及预后间的关系； 3、应用免疫组织化学二步法,检测VEGF、Flt-1和Flk-1在距LPPCN及VM周围最近距离(1-3层)、中等距离(4-6层)及远距离(7-9层)的肿瘤细胞中的表达情况,以及侵袭与转移相关蛋白Twist1、MMP-2、MMP-9在LPPCN阳性组和LPPCN阴性组、VM阳性组和VM阴性组中的表达情况和他们在LPPCN周围肿瘤细胞中的表达情况,并进一步分析VEGF与这些蛋白的表达相关性和它们在VM形成过程中所起的作用。结果 1、黑色素瘤组织中存在LPPCN及VM 对70例黑色素瘤组织进行HE染色可观察到其中有38例瘤组织中出现了胞质浓缩、胞核深染、细胞之间界限清楚的肿瘤细胞,呈线状或网络状分布,此即为LPPCN阳性。对70例黑色素瘤组织进行CD31/PAS双重染色可观察到其中有33例瘤组织中存在一些由CD31染色阴性的肿瘤细胞围成的管腔结构,其管腔壁上有PAS阳性物质,管腔内存在红细胞,周围没有出血坏死和炎症细胞浸润,此即为VM阳性。 2、LPPCN、VM与黑色素瘤患者临床病理参数及预后的关系 LPPCN和VM的发生与患者肿瘤体积的大小有关(P0.05)而与患者的性别、年龄、肿瘤生长部位、有无复发与转移无关(P0.05)。当肿瘤体积大于本实验中肿瘤体积的中位值20cm3的时候较易发生LPPCN和VM(P0.05)。患者的生存率在LPPCN阳性组低于LPPCN阴性组(P0.05),VM阳性组患者的生存率比VM阴性组低(P0.05)。 3、VEGF、Flt-1及Flk-1在LPPCN和VM周围肿瘤细胞中的表达及与预后的关系 VEGF在LPPCN周围肿瘤细胞1-3层中的表达强于其外围肿瘤细胞(P0.05),在外围4-6层与7-9层之间的表达差异无统计学意义(P0.05)。F1k-1在LPPCN周围肿瘤细胞1-3层中的表达强于其外围肿瘤细胞(P0.05),在外围4-6层与7-9层之间的表达差异无统计学意义(P0.05)。而Flt-1在LPPCN周围肿瘤细胞1-3层、4-6层及7-9层之间的表达差异均无统计学意义。VEGF、Flt-1、Flk-1在VM周围肿瘤细胞1-3层、4-6层及7-9层之间的表达差异均无统计学意义(P0.05)。Log-rank检验法比较显示在LPPCN周边VEGF阳性组患者的生存时间比LPPCN周边VEGF阴性组短,差异有统汁学意义(P0.05)。LPPCN周边VEGF阳性组中,VM的阳性率高于LPPCN周边VEGF阴性组中VM的阳性率,差异有统计学意义(P0.05)。 4、侵袭与转移相关蛋白Twist1、MMP-2、MMP-9的表达及与VEGF的相关性 Twist1蛋白在LPPCN阳性组中的表达高于LPPCN阴性组中的表达(P0.05),其在VM阳性组中的表达也高于VM阴性组中的表达(P0.05)。MMP-2蛋白在LPPCN阳性组中的表达高于LPPCN阴性组中的表达(P0.05),其在VM阳性组中的表达也高于VM阴性组中的表达(P0.05)。而MMP-9蛋白在LPPCN阳性组中的表达与LPPCN阴性组中的表达差异比较无统计学意义(P0.05),但在VM阳性组中的表达高于VM阴性组中的表达(P0.05)。相关性分析结果显示,VEGF与MMP-2呈正相关关系(r=0.334,P=0.024); VEGF与MMP-9呈正相关关系(r=0.118,P=0.037); VEGF与Twist1的表达也呈正相关关系(r=0.526, P=0.003)。结论 1、恶性黑色素瘤中确实存在LPPCN和VM,存在VM或/和LPPCN的黑色素瘤患者临床预后差。 2、LPPCN与黑色素瘤中的VM及内皮依赖性血管的形成有密切的关系,LPPCN区瘤细胞消融后形成的空间框架可能是VM及内皮依赖性血管形成的空间结构基础。 3、VEGF及其受体Flk-1在黑色索瘤组织中VM形成的早期可能起一定的作用。VEGF及其受体Flk-1的表达与黑色素瘤患者的临床预后有一定的关系。 4、侵袭与转移相关蛋白MMP-2、MMP-9及Twist1均在VM形成过程中起到了一定的作用,它们与VEGF协同参与了VM1的形成过程。
[Abstract]:objective
Whether there is a linear programmed necrosis of human melanoma tissues (linearly patterned programmed cell necrosis, LPPCN) and vasculogenic mimicry (vasculogenic mimicry, VM) and its distribution, analysis of clinical and pathological significance of their research. 70 cases of human melanoma tissue LPPCN and VM positive cases in LPPCN, VM tumor cell vascular endothelial growth factor (VEGF) and its receptor VEGFR1 (Flt-1) and VEGFR2 (Flk-1) expression and interaction of VEGF with the invasion and metastasis related protein, and to explore their roles in tumor blood Guan Shengcheng and the relationship with clinical prognosis.
Method
1, since 1996-2007 years in Tumour Hospital Affiliated to Tianjin Medical University paraffin samples selected according to the requirement of the experiment (after surgical resection, pathological diagnosis and follow-up data integrity) of the malignant melanoma samples were 70 cases, collected from patients with gender, age, location, tumor size, date of surgery, clinical data and follow-up recurrence and metastasis. The clinical and pathological characteristics of patients with melanoma and its relationship with prognosis;
2, the existence of staining were observed in the presence of VM of the 70 cases of melanoma tissue LPPCN and CD31/PAS double staining method using 70 cases of melanoma tissue by HE, and counted under the microscope. The distribution analysis of LPPCN using correlation method, VM and MVD are related to study of LPPCN and VM, vascular endothelium dependent relationship between, and on the basis of further analysis of LPPCN, the relationship between the clinicopathological parameters and prognosis in the presence of VM in patients with melanoma;
3, the detection of VEGF by immunohistochemistry, Flt-1 and Flk-1 in two steps, at a distance of LPPCN and VM around the nearest distance (1-3 layer), middle distance (4-6 layer) and long distance (7-9 layer) expression in tumor cells, and the invasion and metastasis related proteins Twist1, MMP-2, MMP-9 and LPPCN in the positive group the LPPCN negative group, the expression of VM positive group and VM negative group and in their tumor cells around LPPCN in expression, and further analyze the correlation between the expression of VEGF and the protein and their forming process of role in VM.
Result
1, there is LPPCN and VM in the melanoma tissue
HE staining was observed in 38 cases of tumor tissue appeared in the cytoplasm concentrated on 70 cases of melanoma tissue, hyperchromatic nucleus, well-defined tumor cells, linear or network like distribution, this is LPPCN positive. CD31/PAS double staining can be observed in the lumen of the structure of some tumor cells surrounded by CD31 staining was negative in 33 cases of tumor tissue of 70 cases of melanoma tissue, PAS positive substance of the lumen wall, lumen in the red cell memory, no bleeding around the necrosis and infiltration of inflammatory cells, which were positive for VM.
The relationship between 2, LPPCN, VM and the clinicopathological parameters and prognosis of melanoma patients
LPPCN and VM and the occurrence of patients with tumor size (P0.05) and related to age and gender, tumor location, recurrence and metastasis (P0.05). When the value of 20cm3 was more prone to LPPCN and VM tumor volume greater than the volume of the tumor in this experiment (P0.05) survival. The rate of patients in LPPCN positive group was lower than that of LPPCN negative group (P0.05), the survival rate of VM positive patients than in VM negative group (P0.05).
Expression of 3, VEGF, Flt-1 and Flk-1 in tumor cells around LPPCN and VM and their relationship with prognosis
The strong expression of VEGF in tumor cells around LPPCN in the 1-3 layer in the periphery of tumor cells (P0.05), there was no significant difference in expression between peripheral 4-6 and 7-9 layers (P0.05).F1k-1 in high expression of tumor cells around LPPCN in the 1-3 layer in the periphery of tumor cells (P0.05), no significant difference was found in the the expression between peripheral 4-6 and 7-9 layers (P0.05) and Flt-1 in tumor cells around LPPCN 1-3, difference expression between the 4-6 and 7-9 layers were not statistically significant.VEGF, Flt-1, Flk-1 in tumor cells around VM 1-3 layer, 4-6 layer and 7-9 layer between the expression differences were not statistically significant (P0.05).Log-rank test comparison shows that around LPPCN VEGF positive patients survival time than the surrounding LPPCN VEGF negative group, the difference was significant (P0.05) around.LPPCN VEGF positive group, the positive rate of VM was higher than the positive rate of around LPPCN in VEGF negative group VM, The difference was statistically significant (P0.05).
4, the expression of invasion and metastasis related protein Twist1, MMP-2, MMP-9 and the correlation with VEGF
The expression of Twist1 protein in LPPCN positive group was higher than that in the LPPCN negative group (P0.05), its expression in the VM positive group is higher than that in VM negative group (P0.05) on the expression of.MMP-2 protein in LPPCN positive group was higher than that in LPPCN negative group (P0.05) and its expression in VM positive in the group was higher than that in VM negative group (P0.05). There were no statistically significant differences in the expression of MMP-9 protein in LPPCN positive group and LPPCN negative group the expression in (P0.05), but the expression in the VM positive group was higher than that in VM negative group (P0.05).
Correlation analysis showed that VEGF was positively correlated with MMP-2 (r=0.334, P=0.024), VEGF was positively correlated with MMP-9 (r=0.118, P=0.037), and VEGF was positively correlated with Twist1 expression (r=0.526, VEGF).
conclusion
1, LPPCN and VM do exist in malignant melanoma, and the clinical prognosis of the patients with VM or / and LPPCN melanoma is poor.
2, LPPCN is closely related to the formation of VM and endothelium-dependent blood vessels in melanoma. The spatial framework formed after ablation of LPPCN cells may be the spatial structural basis of VM and endothelium-dependent angiogenesis.
3, VEGF and its receptor Flk-1 may play a certain role in the early stage of VM formation in black tumor tissues. The expression of.VEGF and its receptor Flk-1 is related to the prognosis of patients with melanoma.
4, invasion and metastasis related proteins MMP-2, MMP-9 and Twist1 all play a role in the formation of VM, and they participate in the formation process of VM1 in collaboration with VEGF.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R739.5

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