褐藻多糖硫酸酯对小鼠肝癌Hca-F细胞淋巴道转移的抑制作用

发布时间：2018-03-17 15:30

本文选题：褐藻多糖硫酸酯　切入点：淋巴道转移　出处：《大连医科大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的：肿瘤转移是恶性肿瘤的基本特征和重要标志,肿瘤转移是一个多因素,多步骤,多基因共同调控的复杂过程,涉及大量相关基因结构和表达调控的改变,肿瘤发生转移与肿瘤细胞增殖、粘附、降解细胞外基质、侵袭、迁移密切相关。多糖是一类天然高分子产物,广泛存在于动物细胞膜、植物和微生物细胞壁中,具有多重功效,且毒副作用小。褐藻多糖硫酸酯(Fucoidan)是一类富含L-岩藻糖和硫酸基团的杂多糖,存在于海洋褐藻和棘皮动物体内,具有免疫调节、抗肿瘤i降血、抗衰老等生物活性。其中Fucoidan抗肿瘤活性成为多糖抗肿瘤研究的热点。小鼠肝癌Hca-F和Hca-P细胞系是我校病理学教研室凌茂英教授从近交系昆明小鼠腹水型肝癌细胞H22中筛选建系,其中Hca-F细胞淋巴道转移率高达70%,Hca-P细胞则低于30%,是研究肿瘤淋巴道转移经典细胞模型。血管内皮生长因子-C (VEGF-C)与肿瘤细胞生长、增殖、转移密切相关,VEGFR-3是VEGF-C特异性受体,在某些肿瘤细胞中有表达。本课题组前期研究表明VEGFR-3在Hca-F细胞中有表达。VEGF-C与VEGFR-3结合,可上调肝细胞生长因子(HGF)表达,激活下游信号通路,调控肿瘤的增殖、侵袭转移。肿瘤转移主要分为血道转移和淋巴道转移,淋巴道转移是恶性肿瘤早期发生发展的关键。肿瘤转移首先需要肿瘤细胞自身增殖,进而发生粘附,然后降解细胞外基质,从而发生转移。基质金属蛋白酶家族(MMPs)是降解细胞外基质的主要成员,在促进肿瘤转移过程中具有重要作用,而基质金属蛋白酶组织抑制剂(TIMPs)可以抑制MMPs活性,抑制肿瘤细胞转移能力。本研究以小鼠肝癌Hca-F细胞为细胞模型,采用细胞生物学,生物化学以及分子生物学等方法,探究N-Fucoidan对Hca-F细胞生长、转移、侵袭能力的改变进而探究N-Fucoidan抑制肿瘤转移的分子机制。方法：1.采用乙醇分级沉淀法从粗品Fucoidan中纯化获得N-Fucoidan,使用过氧化氢抗坏血酸联合作用法降解N-Fucoidan,乙醇沉淀后获得LMW-Fucoidan.2.采用苯酚-硫酸法、氯化钡明胶比浊法、硫酸-咔唑法、自动指示旋光仪、核磁共振仪分别测定N-Fucoidan与LMW-Fucoidan的总糖含量、硫酸根含量、糖醛酸含量、旋光度和官能团。3.采用MTT法,Transwell法,Elisa双抗夹心法比较Hca-P细胞和Hca-F细胞生长、侵袭能力、生长因子和MMPs分泌的差异。4.通过台盼蓝染色法检测N-Fucoidan对Hca-F细胞毒作用,并计算存活率。5.MTT法检测N-Fucoidan对Hca-F细胞生长能力的影响,采用Transwell实验检测N-Fucoidan对Hca-F细胞侵袭能力的影响。6. Western blot法检测经过N-Fucoidan处理后Hca-F细胞后,细胞总蛋白中VEGFR-3, C-MET,CXCR-4, TIMP-1蛋白水平表达的影响,并通过ELISA法检测细胞上清中MMP-2、MMP-9、VEGF-C和HGF分泌能力的变化,RT-PCR检测vegf-c, c-met, timp-1相关基因mRNA表达水平的影响。7.MTT法比较N-Fucoidan和LMW-Fucoidan对Hca-F细胞生长能力的影响,ELISA法比对N-Fucoidan和LMW-Fucoidan对VEGF-C、HGF、MMP-2、MMP-9表达水平的不同,Western blot法检测N-Fucoidan和LMW-Fucoidan处理Hca-F细胞后总蛋白中VEGFR-3, C-MET,CXCR-4, TIMP-1蛋白水平表达的影响。8.采用经典淋巴道转移模型,对615小鼠足垫接种Hca-F细胞,N-Fucoidan对体内肿瘤转移的影响。通过ELISA法检测血清中VEGF-C, HGF的含量,HE染色法观察形态变化,免疫组化法检测淋巴管密度。9.采用不完全弗式佐剂诱导mLEC, MTT法检测N-Fucoidan对mLEC细胞增殖能力的影响,免疫荧光法鉴定]mLEC细胞中LYVE-1蛋白的表达,镜下观察N-Fucoidan对]mLEC成管的影响。结果：1. N-Fucoidan的得率为38.91%,从纯化的N-Fucoidan采用过氧化氢-抗坏血酸法降解得到LMW-Fucoidan的得率为60%2. N-Fucoidan总糖含量、硫酸基团含量、糖醛酸含量分别为44.7%、18.5%、12.5%。LMW-Fucoidan总糖含量为48.7%,硫酸基团含量为14.5%、糖醛酸含量为16.5%。3.实验结果表明,Hca-F细胞生长能力是Hca-P细胞的1.79倍,Hca-F细胞侵袭能力是Hca-P细胞的1.67倍,Hca-F细胞分泌VEGF-C、HGF、MMP-2、MMP-9能力分别是Hca-P细胞分泌能力的1.09倍、1.03倍、1.21倍、1.25倍。4. N-Fucoidan处理Hca-F细胞后,细胞死亡率小于1%,表明Fucoidan抑制Hca-F细胞增殖并非细胞毒作用。5.实验结果显示经过N-Fucoidan处理后细胞生长能力收到抑制呈药物浓度及作用时间依赖性关系。Transwell实验结果显示,N-Fucoidan抑制Hca-F细胞发生细胞侵袭,呈时间剂量依赖性关系。6.实验结果表明,N-Fucoidan处理Hca-F细胞后,细胞总蛋白中VEGFR-3、 C-MET、CXCR-4蛋白表达受到抑制,TIMP-1的表达能力被激活。ELISA结果显示；经过N-Fucoidan处理后,N-Fucoidan抑制细胞分泌MMP-2、MMP-9、VEGF-C、 HGF、SDF-1相关生长因子,vegf-c、c-met、vegfr-3相关基因mRNA水平表达下调,timp-1的表达上调。7.实验结果表明LMW-Fucoidan比N-Fucoidan对Hca-F细胞生长具有更强的抑制作用。ELISA结果显示经过药物处理后,Hca-F细胞分泌MMP-2、MMP-9、 VEGF-C、HGF水平及mRNA水平均下调,N-Fucoidan效果强于LMW-Fucoidan。8. N-Fucoidan能够抑制肿瘤细胞生长,增加小鼠脾脏指数和胸腺指数,血清中VEGF-C和HGF的含量下降,肿瘤组织淋巴管密度降低。9.mLEC诱导成功,LYVE-1阳性细胞数大于85%, N-Fucoidan对mLEC生长有抑制作用,呈浓度依赖关系。结论：N-Fucoidan和LMW-Fucoidan对Hca-F细胞生长、侵袭和转移能力均有抑制作用。N-Fucoidan的抑制作用机制与降低MMP-2, MMP-9活性,钝化VEGF-C/VEGFR-3, HGF/C-MET, SDF-1/CXCR-4信号轴通路,降低肿瘤细胞活性及转移能力。
[Abstract]:Objective: tumor metastasis is the basic characteristic of malignant tumor and tumor metastasis is an important symbol of a multi factor, multi steps, complex process regulated many genes, involving a large number of genes changed in structure and expression, metastasis and tumor cell proliferation, tumor adhesion, extracellular matrix degradation, invasion, migration and close. Polysaccharide is a kind of natural polymer product, widely exists in animal cell membranes, plants and microorganisms in the cell wall, has multiple effects and low toxicity. Fucoidan (Fucoidan) is a kind of rich in L- fucose and sulfate radical polysaccharide in marine brown algae and spine the animal skin, have immunomodulatory, antitumor I blood, anti-aging bioactivities. The antitumor activity of Fucoidan has become a hot study of antitumor polysaccharide. Mouse Hca-F and Hca-P cells, is our school Department of Pathology Professor Ling Maoying from Kunming inbred mouse ascites hepatoma cell line H22 screening, including Hca-F cell lymphatic metastasis rate is as high as 70%, Hca-P cells were less than 30%, is the study of lymphatic metastasis of tumor cells. The classical model of vascular endothelial growth factor -C (VEGF-C) and tumor growth, cell proliferation, metastasis VEGFR-3, VEGF-C is the specific receptor, in some tumor cells to express. Previous study showed that the expression of VEGFR-3.VEGF-C combined with VEGFR-3 in Hca-F cells, regulation of hepatocyte growth factor (HGF) expression and activation of downstream signaling pathways, regulation of tumor cell proliferation, invasion and metastasis. Tumor metastasis is mainly divided into the blood and lymph node metastasis, lymph node metastasis of malignant tumor is the key to early occurrence and development of tumor metastasis. First tumor cell proliferation, adhesion and degradation of extracellular matrix, and then, from And the occurrence of metastasis. Matrix metalloproteinases (MMPs) is a leading member of the degradation of extracellular matrix, which plays an important role in the process of promoting tumor metastasis, and tissue inhibitor of matrix metalloproteinase (TIMPs) can inhibit the activity of MMPs, inhibit the metastasis of tumor cells. In this study, mouse liver cancer Hca-F cell by cell biology, biochemistry and molecular biology methods, exploring the N-Fucoidan growth, metastasis of Hca-F cells, molecular mechanism of invasion ability changes and explore the N-Fucoidan inhibition of tumor metastasis. Methods: 1. using ethanol precipitation method from crude and purified Fucoidan in N-Fucoidan, the combined effects of the use of hydrogen peroxide ascorbic acid degradation by N-Fucoidan, ethanol precipitation obtained by LMW-Fucoidan.2. phenol sulfuric acid method, barium chloride gelatin nephelometry carzapol sulfuric acid method, indicating automatic polarimeter, NMR The total sugar content were measured by N-Fucoidan and LMW-Fucoidan NMR, sulfate content, uronic acid content, rotation and.3. groups with MTT method, Transwell method, Elisa double sandwich method between Hca-P cells and Hca-F cell growth, invasion, growth difference factor and MMPs secretion of.4. was detected by trypan N-Fucoidan on Hca-F the cytotoxic effect of blue staining, and calculate the influence survival rate of.5.MTT N-Fucoidan was detected on the proliferation of Hca-F cells were detected by Transwell, the influence of N-Fucoidan on the invasion ability of Hca-F cells.6. Western blot assay after treatment with N-Fucoidan in Hca-F cells, VEGFR-3 cells, total protein in C-MET, CXCR-4, TIMP-1 protein expression level and, by MMP-2, the cell supernatant was detected by ELISA MMP-9, VEGF-C and HGF changes in the secretion of RT-PCR, c-Met, TIMP-1, VEGF-C detection, mRNA gene expression Effect of.7.MTT N-Fucoidan and LMW-Fucoidan flat effect on growth of Hca-F cells, ELISA N-Fucoidan and LMW-Fucoidan method comparison of VEGF-C, HGF, MMP-2, MMP-9 different levels of expression, Western blot method to detect N-Fucoidan and LMW-Fucoidan protein in Hca-F cells after treatment of total VEGFR-3, C-MET, CXCR-4,.8. expression level of TIMP-1 protein by the classic model of lymphatic metastasis, the 615 foot pads of mice inoculated with Hca-F cells, the effect of N-Fucoidan on tumor metastasis. Serum ELISA was detected by VEGF-C method, the content of HGF, observe the morphological changes of HE staining, immunohistochemical detection of lymphatic microvessel density.9. with complete Freund's adjuvant induced mLEC effect detection of N-Fucoidan MTT methods the proliferation of mLEC cells, the expression of LYVE-1 protein by fluorescence method in identification of]mLEC cells, N-Fucoidan tube effect on]mLEC was observed under microscope: 1. N-Fucoidan was 38.91%. The purified N-Fucoidan using hydrogen peroxide ascorbic acid degradation by LMW-Fucoidan was 60%2. N-Fucoidan total sugar content, sulfate content and uronic acid content were 44.7%, 18.5%, the total sugar content of 12.5%.LMW-Fucoidan is 48.7%, the sulfate content was 14.5%, uronic acid content is 16.5%.3. the experimental results show that Hca-F, cell growth ability is 1.79 times that of Hca-P cells, Hca-F cell invasion ability is 1.67 times that of Hca-P cells, Hca-F cells secrete VEGF-C, HGF, MMP-2, MMP-9 were 1.09 times, Hca-P cells secreting capacity of 1.03 times, 1.21 times, 1.25 times.4. N-Fucoidan after treatment of Hca-F cells, cell death rate is less than 1% that showed that Fucoidan inhibited the proliferation of Hca-F cells is not the cytotoxic effect of.5. experimental results showed that after treatment with N-Fucoidan inhibited cell growth ability in drug concentration and action time Dependence of the.Transwell results showed that N-Fucoidan inhibited cell invasion of Hca-F cells in a time dose dependent relationship between the.6. and the experimental results show that N-Fucoidan after treatment of Hca-F cells, VEGFR-3 cells, total protein C-MET, CXCR-4 protein expression was inhibited, the expression ability of TIMP-1 activated by.ELISA results; after N-Fucoidan treatment, the inhibition of N-Fucoidan the secretion of MMP-2 cells, MMP-9, VEGF-C, HGF, SDF-1 VEGF-C, c-Met, growth factor, VEGFR-3 related gene mRNA expression level of TIMP-1, while the expression of.7. test results showed that the inhibitory effect of.ELISA LMW-Fucoidan is stronger than N-Fucoidan on the growth of Hca-F cells showed that after drug treatment, Hca-F cells secrete MMP-2, MMP-9, VEGF-C HGF, and mRNA level declined, N-Fucoidan is better than LMW-Fucoidan.8. N-Fucoidan can inhibit the growth of tumor cells, Increase the spleen index and thymus index of mice, decreased the contents of VEGF-C and HGF in serum, tumor lymphatic density decreased.9.mLEC induced successfully, the number of LYVE-1 positive cells was greater than 85%, N-Fucoidan inhibited the proliferation of mLEC cells in a dose-dependent manner. Conclusion: N-Fucoidan and LMW-Fucoidan on the growth of Hca-F cells, the mechanism of inhibition of invasion and metastasis have the inhibitory effect of.N-Fucoidan and MMP-2 decreased, the activity of MMP-9, VEGF-C/VEGFR-3 HGF/C-MET, SDF-1/CXCR-4 passivation, signal pathway, reduce the activity of tumor cells and metastasis.

【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R73-3

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