黑色素瘤中线形程序性坏死与血管新生及预后关系的初步研究

发布时间：2018-03-21 12:38

本文选题：黑色素瘤　切入点：线形程序性坏死　出处：《天津医科大学》2010年硕士论文　论文类型：学位论文

【摘要】： 目的研究恶性黑色素瘤组织中线形程序性坏死(linearly patterned programmed cell necrosis, LPPCN)的分布及其形态学特点,并探讨其临床病理意义。初步分析LPPCN发生的相关分子机制及其与血管生成拟态(Vasculogenic mimicry, VM)和内皮依赖性血管(Endothelium dependent vessel, EDV)形成的相关性。方法 1.应用形态学分析方法观察恶性黑色素瘤中LPPCN的存在及其分布特点,并探讨其临床病理意义。自1996-2007年天津医科大学附属肿瘤医院存档石蜡标本中,挑选出经手术切除的、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,每例标本均进行HE切片后由有经验的病理医师核实临床病理诊断,进一步观察LPPCN的形态特点并分析LPPCN的分布和临床病理意义。 2.采用免疫组织化学方法初步分析黑色素瘤中LPPCN的发生机制及其与血管新生的相关性。检测黑色素瘤组织中Ⅲ型磷脂酰肌醇三磷酸激酶(ClassⅢPI3K)的表达情况,分析LPPCN与自噬的相关性。检测组织中基质金属蛋白酶2、9(Matrix metalloproteinase2、9, MMP2、MMP9)、金属蛋白酶组织抑制因子(TIMP-1)、组织蛋白酶D (CathepsinD, CathD)、CD105、TGFβ1、p-paxillin、Ⅷ因子相关抗原(Ⅷ-R Ag)及D2-40等蛋白的表达情况,同时进行CD31/PAS双重染色计数血管生成拟态密度(Vasculogenic mimicry density, VMD),从而分析LPPCN与VM、内皮依赖性血管及淋巴管形成的相关性。 3.利用实时荧光定量PCR (Real-time PCR)检测LPPCN阳性组与阴性组中相关基因mRNA的表达差异,分析它们与LPPCN的关系。收集天津医科大学附属肿瘤医院组织库2005年至今新鲜冻存标本黑色素瘤组织30例,通过冰冻切片HE染色观察其中是否存在LPPCN,将其分为LPPCN阳性组与LPPCN阴性组。利用Real-time PCR检测两组中目的基因ClassⅢPI3K、TGFβ1的表达差异,从mRNA的水平分析LPPCN的发生机制及其与血管生成的相关性。结果 1.黑色素瘤中LPPCN的形态学分布特点及其临床病理意义黑色素瘤标本中LPPCN阳性率为54.29%(38/70),LPPCN在HE镜下形态为一簇胞质浓缩、胞核深染的肿瘤细胞,呈线形或网状分布。LPPCN阳性率在不同的肿瘤大小、核分裂像数目及breslow厚度之间差别均具有统计学意义,而与性别、年龄、部位、有无瘤栓、淋巴结转移及复发与远处转移均无关。患者的生存率在LPPCN阳性组低于阴性组(P=0.046)。 2.免疫组化染色结果分析在黑色素瘤组织中,LPPCN阳性组ClassⅢPI3K、CD105标记的微血管密度(microvessel density, MVD)、TGFβ1、Ⅷ-R Ag-MVD、VMD及MMP2表达高于LPPCN阴性组,差别均具有统计学意义(P0.05)；而D2-40标记的微淋巴管密度(lymphvessel density, LVD)、MMP9、CathD在两组间表达差异无统计学意义(P＞0.05); ClassⅢPI3K、TGFβ1在发生LPPCN的肿瘤细胞中的表达高于周围的肿瘤细胞,差异具有统计学意义(P0.05)。CD105与TGFβ1的表达呈正相关；CD105-MVD、Ⅶ-R Ag-MVD及VMD均与LPPCN的密度呈正相关。p-paxillin在黑色素瘤组织中呈普遍低或无表达,也可见其在部分血管内皮细胞中表达。 VM阳性组MMP2、MMP9表达高于VM阴性组,差异具有统计学意义(P0.05)；且TIMP-1在VM阳性组的表达低于VM阴性组,差异具有统计学意义(P＜0.05); CathD在两组间差异无统计学意义(P0.05)。同时本实验结果提示D2-40标记的LVD在淋巴结转移阳性组高于阴性组,差别均具有统计学意义(P0.05)。 3. Real-time PCR结果分析 Real-time PCR结果显示,在恶性黑色素瘤组织中,LPPCN阳性组中ClassⅢPI3K、TGFβ1mRNA表达均高于LPPCN阴性组,与免疫组化结果趋势一致,但仅TGFβ1mRNA表达量在两组间的表达差异有统计学意义(P0.05)。结论 1.恶性黑色素瘤中存在LPPCN及VM,具有LPPCN及VM的患者预后差。 2. LPPCN可能为肿瘤细胞发生的自噬现象,其与黑色素瘤中VM及内皮依赖性血管的形成关系密切,其机制可能是肿瘤细胞发生LPPCN可为血管新生提供—定的空间结构基础。 3.本研究未显示LPPCN与淋巴管生成的相关性。D2-40作为一种新的特异性较强的淋巴管内皮标志物,其与黑色素瘤患者淋巴结转移密切相关。
[Abstract]:objective
Linear necroptosis study of malignant melanoma tissues (linearly patterned programmed cell necrosis, LPPCN) the distribution and morphological characteristics, and explore its clinicopathological significance. Preliminary analysis of the molecular mechanism of the occurrence of LPPCN and vasculogenic mimicry (Vasculogenic mimicry, VM) and endothelium dependent vasodilatation (Endothelium dependent, vessel, EDV) the formation of the relationship.
Method
1. the existence and distribution of LPPCN in malignant melanoma were observed by morphologic analysis, and its clinicopathological significance was discussed.
Since 1996-2007 years in Tumour Hospital Affiliated to Tianjin Medical University paraffin samples, selected by surgical resection, pathological diagnosis and complete follow-up data of malignant melanoma samples were 70 cases, each specimens were sectioned by HE pathology experienced pathologists to verify the clinical diagnosis, to observe the distribution of LPPCN and LPPCN and the analysis of morphological characteristics the clinical and pathological significance.
2. the mechanism of LPPCN in melanoma and its correlation with angiogenesis were preliminarily analyzed by immunohistochemical method.
Detection of type III melanoma tissue phosphatidylinositol three kinase (Class PI3K) expression and correlation analysis of LPPCN and autophagy. Matrix metalloproteinase 2,9 (Matrix detection of metalloproteinase2,9, MMP2, MMP9), tissue inhibitor of metalloproteinase (TIMP-1), cathepsin D (CathepsinD, CathD), CD105 TGF, beta 1, p-paxillin, factor VIII related antigen (VIII -R Ag) expression and D2-40 protein, and CD31/PAS double staining of vasculogenic mimicry density (Vasculogenic mimicry density, VMD, LPPCN and VM) to analyze the correlation, endothelium-dependent and lymphangiogenesis.
3. the difference in the expression of mRNA in LPPCN positive group and negative group was detected by real-time fluorescent quantitative PCR (Real-time PCR), and the relationship between them and LPPCN was analyzed.
The Tumour Hospital Affiliated to Tianjin Medical University tissue bank since 2005 fresh frozen specimens of melanoma tissue in 30 cases, by HE staining observation for the presence of LPPCN, which can be divided into LPPCN positive group and LPPCN negative group. Detected by Real-time PCR two group gene Class III PI3K, the differential expression of TGF beta 1, correlation from mRNA the level of analysis of the mechanism of LPPCN and angiogenesis.
Result
The morphological distribution and clinicopathological significance of LPPCN in 1. melanoma
The positive rate of LPPCN in melanoma specimens was 54.29% (38/70), LPPCN in HE morphology under microscope as a cluster of cytoplasm condensed, deeply stained nuclei of tumor cells, linear or reticular distribution of positive rate of.LPPCN in different tumor size, mitotic differences between the number and the thickness of Breslow was statistically significant, and with gender, age, location, there is no tumor thrombus, lymph node metastasis and recurrence and distant metastasis. The survival rate of the patients in LPPCN positive group was lower than that in negative group (P=0.046).
Analysis of 2. immunohistochemical staining results
In melanoma tissues, LPPCN positive group Class III PI3K, microvessel density marked by CD105 (microvessel density MVD), TGF beta 1, VIII -R Ag-MVD, VMD and MMP2 was higher than that in LPPCN negative group, the difference was statistically significant (P0.05); and the D2-40 markers of lymphatic microvessel density (lymphvessel density LVD, MMP9, CathD), between the two groups in the expression of no significant difference (P > 0.05); Class PI3K, TGF beta 1 in the incidence of LPPCN in tumor cells was higher than that of the surrounding tumor cells, the difference was statistically significant (P0.05) positive expression of.CD105 and TGF beta 1; CD105-MVD, VII -R Ag-MVD and VMD LPPCN and density was positively correlated with.P-paxillin in melanoma tissues was generally low or no expression, which can also be found in some vascular endothelial cells expression.
VM MMP2 positive group, the expression of MMP9 was higher than VM negative group, the difference was statistically significant (P0.05); and TIMP-1 in less than VM negative group the expression of VM positive group, the difference was statistically significant (P < 0.05); there was no significant difference of CathD between the two groups (P0.05). At the same time, the results suggest that D2-40 markers LVD in lymph node metastasis positive group than in negative group, the difference was statistically significant (P0.05).
Analysis of 3. Real-time PCR results
Real-time PCR results showed that in malignant melanoma tissues, the expression of Class III PI3K and TGF beta 1mRNA in LPPCN positive group were all higher than those in LPPCN negative group, which was consistent with the results of immunohistochemistry. However, the expression of TGF beta 1mRNA in the two groups was statistically different (P0.05).
conclusion
In 1. malignant melanoma there are LPPCN and VM. The prognosis of patients with LPPCN and VM is poor.
2. LPPCN may be the autophagy phenomenon of tumor cells, which is closely related to the formation of VM and endothelium-dependent blood vessels in melanoma. The mechanism may be that LPPCN in tumor cells can provide a spatial structural basis for angiogenesis.
3., this study did not show the correlation between LPPCN and lymphangiogenesis..D2-40 as a new specific marker of lymphatic endothelial is closely related to lymph node metastasis in patients with melanoma.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R739.5

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