人淋巴管内皮细胞对卵巢癌淋巴结定向高转移细胞分泌蛋白的影响及候选蛋白的分析与验证

发布时间：2019-06-21 03:45

【摘要】：第一章：人淋巴管内皮细胞对卵巢癌淋巴结定向转移细胞分泌蛋白表达的影响目的：本研究以人卵巢浆液性乳头状腺癌细胞系（SKOV3）、淋巴结定向高转移的亚克隆细胞系（SKOV3-PM4）、人淋巴管内皮细胞（HLEC）为研究对象，建立共培养体系，初步探讨人淋巴管内皮细胞对高转移性上皮性卵巢癌细胞分泌蛋白的影响。方法：将细胞按单独和共培养体系分为4组（A： SKOV3， B： SKOV3+HLEC， C： SKOV3-PM4， D：SKOV3-PM4+HLEC），收集各组细胞的培养上清液。采用抗体芯片和iTRAQ-2D-LC-MALDI-TOF/TOF/MS（基质辅助激光解析电离飞行时间质谱技术和相对与绝对定量同位素标记技术）方法检测各组细胞培养基中的蛋白，筛选出差异明显的分泌蛋白，通过gene oncology、DAVID、string、coremine、KEGG PATHWAY等生物信息学软件，对差异蛋白的功能、生物过程、定位、各蛋白之间的相互作用等进行分析，初步筛选出与卵巢癌淋巴结定向转移相关的节点蛋白。结果：（1）抗体芯片结果表明： C组与A组相比，有39个蛋白发生改变，其中Progranulin（GRN）、VEGFA等34个差异蛋白表达上调，SPARC、IGFBP7等5个差异蛋白表达下调；D与C组相比，有41个蛋白发生改变，其中VEGFA、GRN等22个差异蛋白表达上调，SPARC、IGFBP7等19个差异蛋白表达下调。（2）质谱分析鉴定出SKOV3， SKOV3+HLEC， SKOV3-PM4， SKOV3-PM4+HLEC细胞的分泌蛋白均为200多种（置信度大于95％的蛋白点），SKOV3-PM4与SKOV3相比较，SKOV3-PM4培养上清液中存在36个差异细胞因子，其中表达量上调的有22个，表达量下调的有14个；SKOV3-PM4+HLEC与SKOV3-PM4相比较，共培养后存在65个差异细胞因子，表达上调的有37个，表达量下调的有28个。（3）差异蛋白主要涉及物质代谢、酶活性调节、DNA合成、转录和翻译、细胞内离子运输、细胞粘附等生物学过程。通过蛋白互作网络图分析确定SPARC、VEGFA、GRN等与卵巢癌淋巴结转移密切相关。结论：淋巴结定向转移与淋巴管内皮细胞微环境关系密切，共培养后的差异蛋白涉及到基质细胞蛋白、细胞粘附作用因子、白细胞介素、趋化因子及细胞信号转导等方面。第二章：人淋巴管内皮细胞对卵巢癌淋巴结定向转移细胞分泌蛋白影响的候选蛋白验证目的：对蛋白芯片及质谱鉴定出的差异蛋白进行验证，经生物信息学分析后，选取与淋巴结定向转移高度相关的VEGFA、IGFBP7及SPARC，验证其在临床血清标本和卵巢癌不同淋巴结转移潜能细胞的表达情况。方法：采用ELISA方法对卵巢癌患者血清、卵巢良性患者血清、正常体检者血清标本及细胞培养基进行VEGFA、IGFBP7蛋白验证；采用RT-PCR及免疫荧光技术方法验证SPARC在卵巢癌SKOV3细胞、卵巢癌淋巴结定向高转移SKOV3-PM细胞的表达。结果：经ELISA在卵巢癌、良性肿瘤及正常人血清中验证发现VEGFA、IGFBP7差异有统计学意义（P0.05），VEGFA在恶性组表达最高，正常组表达低；IGFBP7的表达在正常组中表达最高，恶性组中表达低。RT-PCR及免疫荧光技术结果显示SPARC在卵巢癌定向高转移细胞中的表达较卵巢癌细胞下调。结论：VEGFA表达上调及IGFBP7、SPARC的表达下调与卵巢癌淋巴结定向高转移密切相关。第三章：肿瘤微环境与肿瘤淋巴结定向转移的研究进展。（文献综述）正常细胞处于一个相对稳定的内环境，按正常的程序进行着增殖、分化、凋亡以及相关因子的分泌和表达，而肿瘤发生、发展则不断打破这一平衡，逐渐形成一个适于自己生长的组织外环境，即肿瘤微环境。而肿瘤微环境中的众多促淋巴管生成因子、炎性条件、组织缺氧、酸性微环境以及间质高压形成等病理生理特性能促进肿瘤淋巴管生成，，进而促进肿瘤淋巴结转移病灶的形成。本文就肿瘤微环境、淋巴管生成与卵巢癌淋巴结转移的关系作一综述。
[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.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.31

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