多不饱和脂肪酸影响结肠癌发生发展的生物学作用机制研究

发布时间：2018-06-05 00:07

本文选题：多不饱和脂肪酸 + 结肠癌　；参考：《浙江大学》2017年博士论文

【摘要】：结肠癌(colorectalcancer,CRC)是常见的消化道恶性肿瘤。在我国,随着生活方式和饮食结构的改变,结肠癌的发病率也呈现逐年上升的趋势。高脂肪、高蛋白和低膳食纤维的饮食习惯是结肠癌发病的危险因素,饮食干预法是预防结肠癌发生的有效方法,研究发现饮食中降低饱和脂肪的摄入提高多不饱和脂肪酸(Polyunsaturated fatty acids,PUFAs)的比例,可以起到降低肿瘤发病的作用。因此,PUFAs对结肠癌的作用机制值得深入探讨。本研究一方面从体外细胞实验角度探讨PUFAs对结肠癌细胞LoVo和RKO的生长抑制、线粒体损伤、脂肪酸代谢等方面的影响;另一方面从体内动物实验角度出发,通过研究膳食长链不饱和脂肪酸对小鼠肠道生理环境的改善研究其对结肠癌的预防作用。体外部分以人结肠癌细胞LoVo和RKO作为研究对象,构建了 PUFAs对两细胞的体外损伤模型,结果表明本实验用到的六种脂肪酸亚油酸(Linoleic acid,LA)、亚麻酸(Alpha-linolenic acid,ALA)、花生四烯酸(Arachidonic acid,AA)、二十碳五烯酸(Eicosapentaenoic acid,EPA)、二十二碳六烯酸(docosahexaenoic acid,DHA)、γ-亚麻酸(γ-linolenic acud,GLA)浓度大于 120 μM均能显著抑制 LoVo和RKO细胞的生长,诱导两细胞凋亡,但对正常细胞HUVEC影响较小,且半分化细胞RKO对PUFAs的敏感性较未分化细胞LoVo大。根据MTT结果,为了保证脂肪酸处理作用合理高效,我们选择ALA(150μM)、EPA(150μM)、DHA(150μM)、LA(150μM)、GLA(300μM)、AA(150μM)、5-FU(100μM)处理 LoVo细胞,以 ALA(140μM)、EPA(120μM)、DHA(120 μM)、LA(120μM)、GLA(200μM)、AA(120μM)、5-FU(0.4μM)处理RKO,建立PUFAs损伤结肠癌细胞体外模型。以此浓度进一步研究PUFAs对结肠癌细胞LoVo和RKO的线粒体损伤、脂肪酸代谢等方面的影响,发现存在如下效果:(1)从线粒体损伤的角度探讨PUFAs对结肠癌细胞的抑制机制。结果表明,n-3(ALA、EPA、DHA)和 n-6(LA、GLA、AA)脂肪酸作用可致 LoVo 和 RKO细胞内活性氧ROS水平和细胞浆内Ca2+水平上升,ATP含量下降,导致Bcl-2家族中Bax/Bcl-2基因表达比上调。进而引起线粒体膜出现小气孔,线粒体膜电位下降,细胞色素C和AIF等蛋白释放到胞质中,最终激活Caspase-9和Caspase-3活性,引发Caspase级联反应导致细胞凋亡。多不饱和脂肪酸对结肠癌细胞线粒体有一定的损伤作用,通过线粒体凋亡途径诱导其走向死亡。(2)研究PUFAs在LoVo和RKO细胞内的生物转化及其代谢产物对两细胞的影响。油红O染色实验表明外源性添加的PUFAs通过细胞膜以脂滴形式储存于细胞质内。通过气相色谱法分析PUFAs作用下结肠癌细胞内的脂肪酸组成,发现PUFAs作用后改变了 LoVo和RKO细胞内的脂肪酸组成,提高不饱和脂肪酸比例,且在两细胞内n-3和n-6 PUFAs之间的转化存在紧密联系。此外,多不饱和脂肪酸可通过抑制结肠癌细胞COX-2、mPGES、PGDS和ALOX5的表达,进而降低脂肪酸代谢产物促炎因子PGE2和LTB4的分泌,促进抗炎因子LXA4的分泌,使癌细胞趋于不利于其生长的抗炎状态。体内部分以C57BL/6小鼠为对象,通过分析富含长链脂肪酸的鱼油、亚麻籽油、葵花籽油对小鼠结肠菌群结构,小鼠肠道主要代谢产物短链脂肪酸、胆汁酸,小鼠结肠组织脂类代谢参数的影响,分析多不饱和脂肪酸在改善肠道生理环境,预防结肠癌发生方面的功效。结果表明膳食长链脂肪酸可激活脂肪酸转运相关基因(PPARα、OSTα)mRNA的表达促进脂肪代谢,鱼油和亚麻籽油能提高结肠组织中n-3/n-6的比值,保护结肠组织免受氧化应激损伤。此外,膳食长链不饱和脂肪酸能改变小鼠肠道菌群结构,鱼油和亚麻籽油处理组在Eubacterium,Novosphingobium,和Hymenobacter上的丰度高于对照组。富含n-6 PUFAs的葵花籽油组的结肠微生物多样性不如富含n-3 PUFAs鱼油组和亚麻籽油组。亚麻籽油、葵花籽油,特别是鱼油中的多不饱和脂肪酸可促进丁酸产生菌Eubacterium的生长,提高短链脂肪酸含量(特别是丁酸含量提高50%),降低肠道pH值,减少次级胆汁酸含量,起到改善肠道环境的作用,对结肠癌的发生有一定的预防作用。
[Abstract]:Colorectalcancer (CRC) is a common malignant tumor of the digestive tract. In China, with the change of lifestyle and diet structure, the incidence of colon cancer is also increasing year by year. The dietary habits of high fat, high protein and low dietary fiber are the risk factors of colon cancer, and diet intervention is the prevention of colon cancer. Effective methods, the study found that the intake of saturated fat in the diet increased the proportion of Polyunsaturated fatty acids (PUFAs), which can play a role in reducing the incidence of tumor. Therefore, the mechanism of PUFAs on colon cancer should be discussed in depth. On the one hand, this study explores PUFAs on colon cancer from the angle of cell experiment in vitro. The effects of LoVo and RKO on growth inhibition, mitochondrial damage, fatty acid metabolism and other aspects. On the other hand, from the perspective of animal experiments in vivo, the preventive effect of dietary long chain unsaturated fatty acids on the intestinal physiological environment of mice is studied. The outside body is divided into human colon cancer cells LoVo and RKO as a study. Objects, the damage model of PUFAs to two cells was constructed. The results showed that the six fatty acids (Linoleic acid, LA), linolenic acid (Alpha-linolenic acid, ALA), peanut four enoic acid (Arachidonic acid, AA), twenty carbon five enoic acid (Eicosapentaenoic acid, twenty-two), twenty-two carbon six enoic acid, gamma - - gamma, were used in the experiment. The concentration of linolenic acid (gamma -linolenic Acud, GLA) more than 120 u M can significantly inhibit the growth of LoVo and RKO cells and induce two cell apoptosis, but it has little effect on normal cell HUVEC, and the sensitivity of RKO to PUFAs in semi differentiated cells is larger than that of non differentiated cells. According to MTT results, we choose ALA to choose ALA. (150 mu M), EPA (150 mu M), DHA (150 mu M), LA (150 mu M), GLA (300 micron), AA (150 mu M), 5-FU (120 mu), 120 mu (200 mu). The effects of mitochondrial damage and fatty acid metabolism have been found as follows: (1) the inhibition mechanism of PUFAs on colon cancer cells from the angle of mitochondrial damage. The results show that the action of n-3 (ALA, EPA, DHA) and n-6 (LA, GLA, AA) can increase the active oxygen ROS level and the intracellular level in the cytoplasm of LoVo and RKO cells. The decrease in quantity resulted in the increased expression of Bax/Bcl-2 gene in the Bcl-2 family, which led to the emergence of small pores in the mitochondrial membrane, the decrease of mitochondrial membrane potential, the release of cytochrome C and AIF and other proteins into the cytoplasm, and finally the activation of Caspase-9 and Caspase-3, resulting in the apoptosis of the Caspase cascade. Polyunsaturated fatty acids were on the colon cancer cell line. (2) study the biological transformation of PUFAs in LoVo and RKO cells and the effect of its metabolites on two cells. The oil red O staining experiment showed that the exogenous PUFAs was stored in the cytoplasm by the form of lipid droplets in the cell membrane. The analysis of P by gas chromatography UFAs acts as a fatty acid in colon cancer cells. It is found that PUFAs changes the composition of fatty acids in LoVo and RKO cells and increases the proportion of unsaturated fatty acids, and there is a close relationship between the transformation of n-3 and n-6 PUFAs in two cells. In addition, polyunsaturated fatty acids can be used to inhibit colon cancer cells COX-2, mPGES, PGDS, and ALOX5. The expression of fatty acid metabolites, the secretion of pro-inflammatory factors PGE2 and LTB4, the secretion of anti-inflammatory factor LXA4, and the anti inflammatory state of the cancer cells which tend to detrimental to its growth. The body part of the body is in C57BL/6 mice, by analyzing the fish oil rich in long chain fatty acids, subflax seed oil, and sunflower seed oil to the colonic flora of mice. The effects of the main metabolites of the intestinal tract, short chain fatty acids, bile acids, and the lipid metabolic parameters of the colon tissue in mice, were used to analyze the effects of polyunsaturated fatty acids on the improvement of the intestinal physiological environment and the prevention of colon cancer. The results showed that the expression of PPAR alpha, OST alpha by dietary long chain fatty acids activates the expression of fatty acid (mRNA). Metabolism, fish oil and linseed oil can increase the ratio of n-3/n-6 in colon tissue and protect colon tissue from oxidative stress. In addition, dietary long chain unsaturated fatty acids can change the structure of intestinal flora in mice. The abundance of fish oil and linseed oil treatment group on Eubacterium, Novosphingobium, and Hymenobacter is higher than that of the control group. N-6 PU is rich in n-6 PU. The diversity of colonic microorganism in the sunflower seed oil group of FAs is not as good as that of the rich n-3 PUFAs fish oil group and the linseed oil group. The linseed oil, sunflower seed oil, especially the polyunsaturated fatty acids in the fish oil can promote the growth of the butyric acid producing bacteria Eubacterium, increase the content of the short chain fatty acid (especially the butyric acid content by 50%), reduce the pH value of the intestinal tract, and reduce the times. The level of bile acids plays a role in improving the intestinal environment, and has a certain preventive effect on the occurrence of colon cancer.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.35

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