【作者】 卢晓凤；

【导师】 何国庆； 沈生荣；

【作者基本信息】 浙江大学 ， 食品科学， 2011， 博士

【摘要】 随着生活水平的提高以及生活方式的改变,肿瘤的发病率在全球范围内都呈现出上升的趋势,特别是随着人们膳食结构的改变和工作节奏的加快,消化道肿瘤的发病率不断上升。有研究表明改变膳食中脂肪酸的构成,增加多不饱和脂肪酸,可以起到降低肿瘤发病的几率。多不饱和脂肪酸的摄入在对肿瘤的治疗和预后防复发方面也有积极的作用。本文以亚油酸为研究对象,分析了多不饱和脂肪酸在肿瘤细胞及正常细胞中的代谢方式,试图明确其选择性损伤细胞的机理,并探讨其在不同分化状态的细胞中代谢的差异。亚油酸进入细胞后,对细胞生理活性的调节主要存在以下几条途径：1)以膜磷脂的形式贮存于细胞膜中,调节细胞膜的活性；2)作为能量供应物质,经线粒体氧化磷酸化,代谢生成ATP,参与细胞能量代谢；3)可在脂肪酸去饱和酶、延长酶等的作用下代谢生成长链n-6多不饱和脂肪酸,如DGLA,AA等；4)经COX途径或LOX途径相关酶的作用,转化生成不同的信号分子,调节细胞的生存及相关代谢。这几条途径在细胞内共存,并相互影响。本论文以两株不同分化状态的结肠癌细胞及一株正常细胞株为研究对象,重点探讨了不同方式亚油酸处理在不同分化状态细胞株中的毒性及代谢差异,以期为亚油酸在肿瘤防治中的应用提供参考。本研究表明,亚油酸处理可以影响细胞脂肪酸组成,增加细胞不饱和脂肪酸的比例,从而增加细胞膜的流动性,并可能会通过影响细胞脂质筏的正常构象而引起细胞膜通透性的增加,可利用亚油酸的这一特性作为肿瘤治疗的辅助性药物。亚油酸处理可以影响细胞中脂肪酸去饱和酶和延长酶的表达/活性,细胞中长链脂肪酸的比例上升,尽管细胞中脂肪酸的氧化与延长并存,某些细胞中单不饱和脂肪酸的比例有所上升,但细胞中n-6不饱和脂肪酸的比例仍显著性上升,n-6/n-3的比值显著上升。正常处理组的各株细胞存活率与Δn-6/n-3之间呈反比关系,但预处理后的LoVo不再受细胞中n-6多不饱和脂肪酸比例上升所引起的细胞毒性的影响。我们推测这可能是由于不同细胞株对亚油酸诱导产生的细胞毒性的适应性之间存在差异,即不同膳食结构可能对肿瘤细胞亚油酸敏感性之间存在差异。亚油酸处理会影响细胞的自由基变化,从而引起细胞中过氧化现象的发生以及抗氧化能力的变化,但是不同细胞株间存在显著的差异,这些差异通过不同的通路引起细胞线粒体损伤及线粒体细胞色素C的释放,诱导细胞Caspase通路凋亡的发生。但是脂肪酸的处理方式和肿瘤的分化状态会影响细胞对亚油酸的敏感性,经亚油酸预处理后的LoVo细胞株可通过抑制Caspase-3活性抑制凋亡的发生。本研究通过对细胞能荷的分析发现亚油酸处理会影响细胞正常的能量代谢,可通过下调PPARδ的表达而下调脂肪酸的能量代谢。我们同时发现亚油酸会上调细胞中SREBP-2的表达,将细胞氧化的部分能量转化为高能化合物进行贮存。由于SREBP-2可补偿由脂肪酸引起的SREBP-1表达下调,可在一定程度上促进细胞的脂肪酸合成,这也许是细胞中长链脂肪酸比例增加的原因之一。故通过抑制PPARδ以及SREBP-2的表达调节细胞的能量代谢,可能为多不饱和脂肪酸在肿瘤辅助治疗方面的研究提供一定的思路。综上所述,亚油酸诱导细胞损伤的过程与细胞的线粒体损伤和细胞膜损伤均有一定的关系,同时也会在一定程度上影响细胞的正常能量代谢。肿瘤细胞的凋亡是一个复杂的网络调控系统,而亚油酸在肿瘤细胞中所表现出来的毒性也是多方面的。更多还原

【Abstract】 It is reported that the incidence of cancer worldwild is increasing rapidly in recent years with the promotion of living condition and life style. The incidence of gastrointestinal cancer is rising with the change of diet pattern and lack of exercise. Researches had proved that changes in diet fatty acids composition, raising the ratio of polyunsaturated fatty acids would help in reducing the incidence of gastrointestinal tumors, especially the n-3 polyunsaturated fatty acids. Polyunsaturated fatty acids could also help in tumor cure and prevention of tumor recurrence. In the present work, we analyzed the metabolic pathway of linoleic acid in two colorectural tumor cell lines and a normal cell line HUVEC taken as control to illustrate the mechanisms of cell apoptosis induced by polyunsaturated fatty acids, and try to find if there were differences between different cell lines with different cell differentiation status.Linoleic acid could regulate cell physiological activity after taken in cell by the following four pathways:i) linoleic acid could be stored in cell membrane as phospholipid to modulate cell membrane activity; ii) linoleic acid could be oxidated in mitochondrial and providing ATP for cell energy metabolism; iii) linoleic acid could be transformed into DGLA or AA with the expression of fatty acids desaturases and enlongases; iv) linoleic acid could be transformed into signal molecules to regulate cell viability by COX pathway or LOX pathway. The four functions of linoleic acid existed in cell at the same time and interacted with each other. In the present research, we discussed the cell sensitivity of linoleic acid with different linoleic acid treatments and different cell differentiation status among two colorectual cancer cell lines, LoVo (undifferentiated) and Rko (semi-differentiatied), and one normal cell line HUVEC (human umbilical vein endothelial cell).Our research proved that linoleic acid could affect the cell fatty acids composition by raising the ratio of polyunsaturated fatty acids, especially the n-6 polyunsaturated fatty acids, which could induce the rise of cell membrane fluidity and finally increase the cell membrane permeability by possibly affecting the normal function of cell lipid rafts. Treatment with linoleic acid could affect the expression/activity of fatty acids desaturases and enlongases, so the ratio of long chain fatty acids to cell total fatty acids raised significantly, likewise the ratios of n-6 polyunsaturated fatty acids and n-6/n-3 in cell, while the oxidation of fatty acids was also progressed and the ratio of monounsaturated fatty acids raised in some treatments. Cell viability had inverse relation toΔn-6/n-3 in normal treated cells with all three cell lines, while LoVo could resist to linoleic acid toxin after pre-treatments. So the bioactivity differences of linoleic acid between cells may related to the differences of cell metabolism pathway, and different diet comsumption may lead to different sensitivity of cells to linoleic acid.Additional linoleic acid may affect the cell by initiating free radicals, as different cells have different ways to reduce the cell concentration of free radicals, both LoVo and Rko could accumulate MDA at a relative high level, Rko could also have a significant change in SOD activity, while HUVEC could tolerate a high concentration of linoleic acid. But high concentration could induce mitochondrial damage to all the three cell lines with cytochrome C release and activity of caspase pathway, while LoVo treated with pre-treatment and then high concentration of linoleic acid could survive by inhibiting the activity of caspase-3. So we concluded that the regulation of caspase signal way could affect the cell apoptosis induced by linoleic acid.By analysis of cell energy charge, we found that linoleic acid could affect the normal cell energy metabolism, and this might be caused by the down-regulation of PPARδwith reducing the metabolic of free fatty acid. Linoleic acid could up-regulate the expression of SREBP-2 as it could storage energy by producing high-energy compounds. The up-regulation of SREBP-2, which could compensate for the SREBP-1 down-regulation, could also help in fatty acids synthesis, which may cause the ratio rise of long chain fatty acids in cell. To block the activity of PPARδand SREBP-2 when the tumor cells exposed to PUFAs may reduce the cell viability by regulating the cell energy charge, this would be another way for exploring polyunsaturated fatty acids’function in inducing tumor cell apoptosis.In summary, linoleic acid could induce tumor cell mitochondrial impairment, cell membrane damage, and to some extent have influence to cell energy change. As the apoptosis of tumor cell is a complicated network, the activity of linoleic acid could also modulate the apoptosis in many ways.更多还原