【摘要】：第一章：人淋巴管內(nèi)皮細(xì)胞對(duì)卵巢癌淋巴結(jié)定向轉(zhuǎn)移細(xì)胞分泌蛋白表達(dá)的影響 目的：本研究以人卵巢漿液性乳頭狀腺癌細(xì)胞系（SKOV3）、淋巴結(jié)定向高轉(zhuǎn)移的亞克隆細(xì)胞系（SKOV3-PM4）、人淋巴管內(nèi)皮細(xì)胞（HLEC）為研究對(duì)象，建立共培養(yǎng)體系，初步探討人淋巴管內(nèi)皮細(xì)胞對(duì)高轉(zhuǎn)移性上皮性卵巢癌細(xì)胞分泌蛋白的影響。方法：將細(xì)胞按單獨(dú)和共培養(yǎng)體系分為4組（A： SKOV3， B： SKOV3+HLEC， C： SKOV3-PM4， D：SKOV3-PM4+HLEC），收集各組細(xì)胞的培養(yǎng)上清液。采用抗體芯片和iTRAQ-2D-LC-MALDI-TOF/TOF/MS（基質(zhì)輔助激光解析電離飛行時(shí)間質(zhì)譜技術(shù)和相對(duì)與絕對(duì)定量同位素標(biāo)記技術(shù)）方法檢測(cè)各組細(xì)胞培養(yǎng)基中的蛋白，篩選出差異明顯的分泌蛋白，通過gene oncology、DAVID、string、coremine、KEGG PATHWAY等生物信息學(xué)軟件，對(duì)差異蛋白的功能、生物過程、定位、各蛋白之間的相互作用等進(jìn)行分析，初步篩選出與卵巢癌淋巴結(jié)定向轉(zhuǎn)移相關(guān)的節(jié)點(diǎn)蛋白。結(jié)果：（1）抗體芯片結(jié)果表明： C組與A組相比，有39個(gè)蛋白發(fā)生改變，其中Progranulin（GRN）、VEGFA等34個(gè)差異蛋白表達(dá)上調(diào)，SPARC、IGFBP7等5個(gè)差異蛋白表達(dá)下調(diào)；D與C組相比，有41個(gè)蛋白發(fā)生改變，其中VEGFA、GRN等22個(gè)差異蛋白表達(dá)上調(diào)，SPARC、IGFBP7等19個(gè)差異蛋白表達(dá)下調(diào)。（2）質(zhì)譜分析鑒定出SKOV3， SKOV3+HLEC， SKOV3-PM4， SKOV3-PM4+HLEC細(xì)胞的分泌蛋白均為200多種（置信度大于95％的蛋白點(diǎn)），SKOV3-PM4與SKOV3相比較，SKOV3-PM4培養(yǎng)上清液中存在36個(gè)差異細(xì)胞因子，其中表達(dá)量上調(diào)的有22個(gè)，表達(dá)量下調(diào)的有14個(gè)；SKOV3-PM4+HLEC與SKOV3-PM4相比較，共培養(yǎng)后存在65個(gè)差異細(xì)胞因子，表達(dá)上調(diào)的有37個(gè)，表達(dá)量下調(diào)的有28個(gè)。（3）差異蛋白主要涉及物質(zhì)代謝、酶活性調(diào)節(jié)、DNA合成、轉(zhuǎn)錄和翻譯、細(xì)胞內(nèi)離子運(yùn)輸、細(xì)胞粘附等生物學(xué)過程。通過蛋白互作網(wǎng)絡(luò)圖分析確定SPARC、VEGFA、GRN等與卵巢癌淋巴結(jié)轉(zhuǎn)移密切相關(guān)。結(jié)論：淋巴結(jié)定向轉(zhuǎn)移與淋巴管內(nèi)皮細(xì)胞微環(huán)境關(guān)系密切，共培養(yǎng)后的差異蛋白涉及到基質(zhì)細(xì)胞蛋白、細(xì)胞粘附作用因子、白細(xì)胞介素、趨化因子及細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)等方面。 第二章：人淋巴管內(nèi)皮細(xì)胞對(duì)卵巢癌淋巴結(jié)定向轉(zhuǎn)移細(xì)胞分泌蛋白影響的候選蛋白驗(yàn)證 目的：對(duì)蛋白芯片及質(zhì)譜鑒定出的差異蛋白進(jìn)行驗(yàn)證，經(jīng)生物信息學(xué)分析后，選取與淋巴結(jié)定向轉(zhuǎn)移高度相關(guān)的VEGFA、IGFBP7及SPARC，驗(yàn)證其在臨床血清標(biāo)本和卵巢癌不同淋巴結(jié)轉(zhuǎn)移潛能細(xì)胞的表達(dá)情況。方法：采用ELISA方法對(duì)卵巢癌患者血清、卵巢良性患者血清、正常體檢者血清標(biāo)本及細(xì)胞培養(yǎng)基進(jìn)行VEGFA、IGFBP7蛋白驗(yàn)證；采用RT-PCR及免疫熒光技術(shù)方法驗(yàn)證SPARC在卵巢癌SKOV3細(xì)胞、卵巢癌淋巴結(jié)定向高轉(zhuǎn)移SKOV3-PM細(xì)胞的表達(dá)。結(jié)果：經(jīng)ELISA在卵巢癌、良性腫瘤及正常人血清中驗(yàn)證發(fā)現(xiàn)VEGFA、IGFBP7差異有統(tǒng)計(jì)學(xué)意義（P0.05），VEGFA在惡性組表達(dá)最高，正常組表達(dá)低；IGFBP7的表達(dá)在正常組中表達(dá)最高，惡性組中表達(dá)低。RT-PCR及免疫熒光技術(shù)結(jié)果顯示SPARC在卵巢癌定向高轉(zhuǎn)移細(xì)胞中的表達(dá)較卵巢癌細(xì)胞下調(diào)。結(jié)論：VEGFA表達(dá)上調(diào)及IGFBP7、SPARC的表達(dá)下調(diào)與卵巢癌淋巴結(jié)定向高轉(zhuǎn)移密切相關(guān)。 第三章：腫瘤微環(huán)境與腫瘤淋巴結(jié)定向轉(zhuǎn)移的研究進(jìn)展。（文獻(xiàn)綜述） 正常細(xì)胞處于一個(gè)相對(duì)穩(wěn)定的內(nèi)環(huán)境，按正常的程序進(jìn)行著增殖、分化、凋亡以及相關(guān)因子的分泌和表達(dá)，而腫瘤發(fā)生、發(fā)展則不斷打破這一平衡，逐漸形成一個(gè)適于自己生長(zhǎng)的組織外環(huán)境，即腫瘤微環(huán)境。而腫瘤微環(huán)境中的眾多促淋巴管生成因子、炎性條件、組織缺氧、酸性微環(huán)境以及間質(zhì)高壓形成等病理生理特性能促進(jìn)腫瘤淋巴管生成，，進(jìn)而促進(jìn)腫瘤淋巴結(jié)轉(zhuǎn)移病灶的形成。本文就腫瘤微環(huán)境、淋巴管生成與卵巢癌淋巴結(jié)轉(zhuǎn)移的關(guān)系作一綜述。
[Abstract]:The first chapter: the effect of human lymphatic endothelial cell on the expression of the secretory protein of the metastatic cell of ovarian cancer Objective: To study the subclone cell line (SKOV3-PM4) with high metastasis of human ovarian serous papillary adenocarcinoma cell line (SKOV3) and lymph node. Objective: To establish a co-culture system for human lymphatic endothelial cells (HLEC) and to explore the effect of human lymphatic endothelial cells on the secretion of highly metastatic epithelial ovarian cancer cells. Methods: The cells were divided into 4 groups (A: SKOV3, B: SKOV3 + HLEC, C: SKOV3-PM4, D: SKOV3-PM4 + HLEC) in a single and co-culture system. Liquid. The protein in each group of cell culture media was tested by using the antibody chip and the iTRAQ-2D-LC-MALDI-TOF/ TOF/ MS (matrix-assisted laser ionization time-of-flight mass spectrometry and relative and absolute quantitative isotope labeling technology). E. Bioinformatics software such as KEGG PATHWAY, the function of the differential protein, the biological process, the location, the interaction among the proteins, and the like are analyzed, and the node egg related to the directional transfer of the ovarian cancer lymph node is preliminarily screened. Results: (1) The results showed that: (1) The results showed that: (1) The results showed that, compared with group A, there were 39 protein changes, among which, the expression of 5 different proteins, such as Propanulin (GRN) and GFA, was down-regulated, and the expression of 5 different proteins, such as SPARC and IGFBP7, was down-regulated; compared with group C, there were 41 protein changes, of which VEGF The expression of 19 differentially expressed proteins, such as A, GRN and the like, was up-regulated, and the expression of 19 differential proteins, such as SPARC, IGFBP7, and the like The secretion of SKOV3, SKOV3 + HLEC, SKOV3-PM4, SKOV3-PM4 + HLEC cells was more than 200 (95% confidence level), and the SKOV3-PM4 was compared with SKOV3. The expression of SKOV3-PM4 + HLEC and SKOV3-PM4 was compared with that of SKOV3-PM4. (3) The differential protein is mainly involved in the biology of substance metabolism, enzyme activity regulation, DNA synthesis, transcription and translation, intracellular ion transport, cell adhesion, etc. Cheng. Based on the analysis of protein interaction network graph, it is determined that SPARC, BFA, GRN and so on are closely related to the lymph node metastasis of ovarian cancer. Conclusion: The lymph node directional metastasis is closely related to the microenvironment of the lymphatic endothelial cells, and the co-cultured differential protein is involved in the matrix cell protein, the cell adhesion factor, the interleukin, the chemokines and the cell signal transduction. The second chapter: the candidate for the effect of human lymphatic endothelial cell on the secretion of the human ovarian cancer lymph node Protein verification purpose: The differential protein identified by the protein chip and the mass spectrum is verified and the biological letter is obtained. EFA, IGFBP7, and SPARC, which were highly correlated with the lymph node directional transfer height, were selected to verify the metastatic potential cells of different lymph nodes in the clinical serum and ovarian cancer. Methods: The serum of the patients with ovarian cancer, the serum of the patients with benign ovarian cancer, the serum samples and the cell culture medium of the patients with ovarian cancer were verified by ELISA. The expression of IGFBP7 was verified by using the method of RT-PCR and immunofluorescence. OV3 cell, high metastatic SKOV3-PM in lymph node of ovarian cancer The results showed that the expression of IGFBP7 was the highest in the normal group and the expression of IGFBP7 was the highest in the normal group, and the expression of IGFBP7 was the highest in the normal group, and the expression of IGFBP7 was the highest in the normal group. The expression of low. RT-PCR and immunofluorescence in the group showed that the expression of SPARC in the highly metastatic cells of ovarian cancer was higher than that in the ovary. Conclusion: The up-regulation of the expression of HFA and the down-regulation of IGFBP7 and SPARC and the high rotation of the lymph node in the ovarian cancer The third chapter: the micro-environment of the tumor and the orientation of the tumor's lymph node The Study of the Transfer (Literature review) The normal cells are in a relatively stable internal environment, proliferation, differentiation, apoptosis and the secretion and expression of the related factors are carried out according to the normal procedure, and the tumorigenesis and development to break this balance and gradually to form an organization that is suitable for its own growth The environment, that is, the micro-environment of the tumor, and many of the lymphangiogenesis factors, the inflammatory conditions, the tissue hypoxia, the acidic microenvironment and the formation of interstitial high-pressure in the micro-environment of the tumor can promote the lymphangiogenesis of the tumor, and further promote the treatment of the tumor. The formation of the metastatic lesions of the Ba-junction. This paper deals with the tumor microenvironment, lymphangiogenesis and the lymph node of ovarian cancer.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.31

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