本文選題：黑色素瘤　切入點：線形程序性壞死　出處：《天津醫(yī)科大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的 研究惡性黑色素瘤組織中線形程序性壞死(linearly patterned programmed cell necrosis, LPPCN)的分布及其形態(tài)學(xué)特點,并探討其臨床病理意義。初步分析LPPCN發(fā)生的相關(guān)分子機制及其與血管生成擬態(tài)(Vasculogenic mimicry, VM)和內(nèi)皮依賴性血管(Endothelium dependent vessel, EDV)形成的相關(guān)性。 方法 1.應(yīng)用形態(tài)學(xué)分析方法觀察惡性黑色素瘤中LPPCN的存在及其分布特點,并探討其臨床病理意義。 自1996-2007年天津醫(yī)科大學(xué)附屬腫瘤醫(yī)院存檔石蠟標(biāo)本中,挑選出經(jīng)手術(shù)切除的、病理診斷明確且隨訪資料完整的惡性黑色素瘤標(biāo)本共70例,每例標(biāo)本均進行HE切片后由有經(jīng)驗的病理醫(yī)師核實臨床病理診斷,進一步觀察LPPCN的形態(tài)特點并分析LPPCN的分布和臨床病理意義。 2.采用免疫組織化學(xué)方法初步分析黑色素瘤中LPPCN的發(fā)生機制及其與血管新生的相關(guān)性。 檢測黑色素瘤組織中Ⅲ型磷脂酰肌醇三磷酸激酶(ClassⅢPI3K)的表達情況,分析LPPCN與自噬的相關(guān)性。檢測組織中基質(zhì)金屬蛋白酶2、9(Matrix metalloproteinase2、9, MMP2、MMP9)、金屬蛋白酶組織抑制因子(TIMP-1)、組織蛋白酶D (CathepsinD, CathD)、CD105、TGFβ1、p-paxillin、Ⅷ因子相關(guān)抗原(Ⅷ-R Ag)及D2-40等蛋白的表達情況,同時進行CD31/PAS雙重染色計數(shù)血管生成擬態(tài)密度(Vasculogenic mimicry density, VMD),從而分析LPPCN與VM、內(nèi)皮依賴性血管及淋巴管形成的相關(guān)性。 3.利用實時熒光定量PCR (Real-time PCR)檢測LPPCN陽性組與陰性組中相關(guān)基因mRNA的表達差異,分析它們與LPPCN的關(guān)系。 收集天津醫(yī)科大學(xué)附屬腫瘤醫(yī)院組織庫2005年至今新鮮凍存標(biāo)本黑色素瘤組織30例,通過冰凍切片HE染色觀察其中是否存在LPPCN,將其分為LPPCN陽性組與LPPCN陰性組。利用Real-time PCR檢測兩組中目的基因ClassⅢPI3K、TGFβ1的表達差異,從mRNA的水平分析LPPCN的發(fā)生機制及其與血管生成的相關(guān)性。 結(jié)果 1.黑色素瘤中LPPCN的形態(tài)學(xué)分布特點及其臨床病理意義 黑色素瘤標(biāo)本中LPPCN陽性率為54.29%(38/70),LPPCN在HE鏡下形態(tài)為一簇胞質(zhì)濃縮、胞核深染的腫瘤細(xì)胞,呈線形或網(wǎng)狀分布。LPPCN陽性率在不同的腫瘤大小、核分裂像數(shù)目及breslow厚度之間差別均具有統(tǒng)計學(xué)意義,而與性別、年齡、部位、有無瘤栓、淋巴結(jié)轉(zhuǎn)移及復(fù)發(fā)與遠處轉(zhuǎn)移均無關(guān)。患者的生存率在LPPCN陽性組低于陰性組(P=0.046)。 2.免疫組化染色結(jié)果分析 在黑色素瘤組織中,LPPCN陽性組ClassⅢPI3K、CD105標(biāo)記的微血管密度(microvessel density, MVD)、TGFβ1、Ⅷ-R Ag-MVD、VMD及MMP2表達高于LPPCN陰性組,差別均具有統(tǒng)計學(xué)意義(P0.05)；而D2-40標(biāo)記的微淋巴管密度(lymphvessel density, LVD)、MMP9、CathD在兩組間表達差異無統(tǒng)計學(xué)意義(P＞0.05); ClassⅢPI3K、TGFβ1在發(fā)生LPPCN的腫瘤細(xì)胞中的表達高于周圍的腫瘤細(xì)胞,差異具有統(tǒng)計學(xué)意義(P0.05)。CD105與TGFβ1的表達呈正相關(guān)；CD105-MVD、Ⅶ-R Ag-MVD及VMD均與LPPCN的密度呈正相關(guān)。p-paxillin在黑色素瘤組織中呈普遍低或無表達,也可見其在部分血管內(nèi)皮細(xì)胞中表達。 VM陽性組MMP2、MMP9表達高于VM陰性組,差異具有統(tǒng)計學(xué)意義(P0.05)；且TIMP-1在VM陽性組的表達低于VM陰性組,差異具有統(tǒng)計學(xué)意義(P＜0.05); CathD在兩組間差異無統(tǒng)計學(xué)意義(P0.05)。同時本實驗結(jié)果提示D2-40標(biāo)記的LVD在淋巴結(jié)轉(zhuǎn)移陽性組高于陰性組,差別均具有統(tǒng)計學(xué)意義(P0.05)。 3. Real-time PCR結(jié)果分析 Real-time PCR結(jié)果顯示,在惡性黑色素瘤組織中,LPPCN陽性組中ClassⅢPI3K、TGFβ1mRNA表達均高于LPPCN陰性組,與免疫組化結(jié)果趨勢一致,但僅TGFβ1mRNA表達量在兩組間的表達差異有統(tǒng)計學(xué)意義(P0.05)。 結(jié)論 1.惡性黑色素瘤中存在LPPCN及VM,具有LPPCN及VM的患者預(yù)后差。 2. LPPCN可能為腫瘤細(xì)胞發(fā)生的自噬現(xiàn)象,其與黑色素瘤中VM及內(nèi)皮依賴性血管的形成關(guān)系密切,其機制可能是腫瘤細(xì)胞發(fā)生LPPCN可為血管新生提供—定的空間結(jié)構(gòu)基礎(chǔ)。 3.本研究未顯示LPPCN與淋巴管生成的相關(guān)性。D2-40作為一種新的特異性較強的淋巴管內(nèi)皮標(biāo)志物,其與黑色素瘤患者淋巴結(jié)轉(zhuǎn)移密切相關(guān)。
[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.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R739.5

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