本文關鍵詞： 黑色素瘤 線形程序性壞死 血管生成擬態(tài) 血管內(nèi)皮生長因子 受體　出處：《天津醫(yī)科大學》2011年碩士論文　論文類型：學位論文

【摘要】：目的 研究人體黑色素瘤組織中是否存在線形程序性壞死(linearly patterned programmed cell necrosis, LPPCN)及血管生成擬態(tài)(vasculogenic mimicry, VM)及其分布情況,分析它們的臨床病理意義。研究70例人體黑色素瘤組織中LPPCN及VM陽性病例中的LPPCN、VM周圍腫瘤細胞血.管內(nèi)皮生長因子(VEGF)及其受體VEGFR1 (Flt-1)和VEGFR2 (Flk-1)的表達情況并研究VEGF與侵襲轉(zhuǎn)移相關蛋白的相互作用,探討它們在腫瘤血管生成中的作用以及與臨床預后的關系。 方法 1、自1996-2007年天津醫(yī)科大學附屬腫瘤醫(yī)院存檔石蠟標本中挑選出符合實驗要求(經(jīng)手術切除、病理診斷明確且隨訪資料完整)的惡性黑色素瘤標本共70例,收集患者的性別、年齡、發(fā)病部位、腫瘤大小、手術日期、復發(fā)與轉(zhuǎn)移和隨訪等臨床資料。分析黑色素瘤患者的臨床病理特征及其與預后的關系； 2、利用HE染色觀察70例黑色素瘤組織中是否存在LPPCN及利用CD31/PAS雙重染色的方法來觀察這70例黑色素瘤組織中是否存在VM,并在顯微鏡下計數(shù)其各自的分布情況。運用相關檢驗法分析LPPCN、VM與MVD之間有無相關以探討LPPCN與VM、內(nèi)皮依賴性血管之間的關聯(lián)性,并在此基礎上進一步分析LPPCN、VM的存在與黑色素瘤患者的臨床病理參數(shù)及預后間的關系； 3、應用免疫組織化學二步法,檢測VEGF、Flt-1和Flk-1在距LPPCN及VM周圍最近距離(1-3層)、中等距離(4-6層)及遠距離(7-9層)的腫瘤細胞中的表達情況,以及侵襲與轉(zhuǎn)移相關蛋白Twist1、MMP-2、MMP-9在LPPCN陽性組和LPPCN陰性組、VM陽性組和VM陰性組中的表達情況和他們在LPPCN周圍腫瘤細胞中的表達情況,并進一步分析VEGF與這些蛋白的表達相關性和它們在VM形成過程中所起的作用。 結(jié)果 1、黑色素瘤組織中存在LPPCN及VM 對70例黑色素瘤組織進行HE染色可觀察到其中有38例瘤組織中出現(xiàn)了胞質(zhì)濃縮、胞核深染、細胞之間界限清楚的腫瘤細胞,呈線狀或網(wǎng)絡狀分布,此即為LPPCN陽性。對70例黑色素瘤組織進行CD31/PAS雙重染色可觀察到其中有33例瘤組織中存在一些由CD31染色陰性的腫瘤細胞圍成的管腔結(jié)構(gòu),其管腔壁上有PAS陽性物質(zhì),管腔內(nèi)存在紅細胞,周圍沒有出血壞死和炎癥細胞浸潤,此即為VM陽性。 2、LPPCN、VM與黑色素瘤患者臨床病理參數(shù)及預后的關系 LPPCN和VM的發(fā)生與患者腫瘤體積的大小有關(P0.05)而與患者的性別、年齡、腫瘤生長部位、有無復發(fā)與轉(zhuǎn)移無關(P0.05)。當腫瘤體積大于本實驗中腫瘤體積的中位值20cm3的時候較易發(fā)生LPPCN和VM(P0.05)�；颊叩纳媛试贚PPCN陽性組低于LPPCN陰性組(P0.05),VM陽性組患者的生存率比VM陰性組低(P0.05)。 3、VEGF、Flt-1及Flk-1在LPPCN和VM周圍腫瘤細胞中的表達及與預后的關系 VEGF在LPPCN周圍腫瘤細胞1-3層中的表達強于其外圍腫瘤細胞(P0.05),在外圍4-6層與7-9層之間的表達差異無統(tǒng)計學意義(P0.05)。F1k-1在LPPCN周圍腫瘤細胞1-3層中的表達強于其外圍腫瘤細胞(P0.05),在外圍4-6層與7-9層之間的表達差異無統(tǒng)計學意義(P0.05)。而Flt-1在LPPCN周圍腫瘤細胞1-3層、4-6層及7-9層之間的表達差異均無統(tǒng)計學意義。VEGF、Flt-1、Flk-1在VM周圍腫瘤細胞1-3層、4-6層及7-9層之間的表達差異均無統(tǒng)計學意義(P0.05)。Log-rank檢驗法比較顯示在LPPCN周邊VEGF陽性組患者的生存時間比LPPCN周邊VEGF陰性組短,差異有統(tǒng)汁學意義(P0.05)。LPPCN周邊VEGF陽性組中,VM的陽性率高于LPPCN周邊VEGF陰性組中VM的陽性率,差異有統(tǒng)計學意義(P0.05)。 4、侵襲與轉(zhuǎn)移相關蛋白Twist1、MMP-2、MMP-9的表達及與VEGF的相關性 Twist1蛋白在LPPCN陽性組中的表達高于LPPCN陰性組中的表達(P0.05),其在VM陽性組中的表達也高于VM陰性組中的表達(P0.05)。MMP-2蛋白在LPPCN陽性組中的表達高于LPPCN陰性組中的表達(P0.05),其在VM陽性組中的表達也高于VM陰性組中的表達(P0.05)。而MMP-9蛋白在LPPCN陽性組中的表達與LPPCN陰性組中的表達差異比較無統(tǒng)計學意義(P0.05),但在VM陽性組中的表達高于VM陰性組中的表達(P0.05)。 相關性分析結(jié)果顯示,VEGF與MMP-2呈正相關關系(r=0.334,P=0.024); VEGF與MMP-9呈正相關關系(r=0.118,P=0.037); VEGF與Twist1的表達也呈正相關關系(r=0.526, P=0.003)。 結(jié)論 1、惡性黑色素瘤中確實存在LPPCN和VM,存在VM或/和LPPCN的黑色素瘤患者臨床預后差。 2、LPPCN與黑色素瘤中的VM及內(nèi)皮依賴性血管的形成有密切的關系,LPPCN區(qū)瘤細胞消融后形成的空間框架可能是VM及內(nèi)皮依賴性血管形成的空間結(jié)構(gòu)基礎。 3、VEGF及其受體Flk-1在黑色索瘤組織中VM形成的早期可能起一定的作用。VEGF及其受體Flk-1的表達與黑色素瘤患者的臨床預后有一定的關系。 4、侵襲與轉(zhuǎn)移相關蛋白MMP-2、MMP-9及Twist1均在VM形成過程中起到了一定的作用,它們與VEGF協(xié)同參與了VM1的形成過程。
[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.

【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R739.5

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