發(fā)布時間：2018-06-05 00:07

  本文選題：多不飽和脂肪酸 + 結(jié)腸癌��； 參考：《浙江大學(xué)》2017年博士論文

【摘要】：結(jié)腸癌(colorectalcancer,CRC)是常見的消化道惡性腫瘤。在我國,隨著生活方式和飲食結(jié)構(gòu)的改變,結(jié)腸癌的發(fā)病率也呈現(xiàn)逐年上升的趨勢。高脂肪、高蛋白和低膳食纖維的飲食習(xí)慣是結(jié)腸癌發(fā)病的危險因素,飲食干預(yù)法是預(yù)防結(jié)腸癌發(fā)生的有效方法,研究發(fā)現(xiàn)飲食中降低飽和脂肪的攝入提高多不飽和脂肪酸(Polyunsaturated fatty acids,PUFAs)的比例,可以起到降低腫瘤發(fā)病的作用。因此,PUFAs對結(jié)腸癌的作用機(jī)制值得深入探討。本研究一方面從體外細(xì)胞實驗角度探討PUFAs對結(jié)腸癌細(xì)胞LoVo和RKO的生長抑制、線粒體損傷、脂肪酸代謝等方面的影響;另一方面從體內(nèi)動物實驗角度出發(fā),通過研究膳食長鏈不飽和脂肪酸對小鼠腸道生理環(huán)境的改善研究其對結(jié)腸癌的預(yù)防作用。體外部分以人結(jié)腸癌細(xì)胞LoVo和RKO作為研究對象,構(gòu)建了 PUFAs對兩細(xì)胞的體外損傷模型,結(jié)果表明本實驗用到的六種脂肪酸亞油酸(Linoleic acid,LA)、亞麻酸(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-亞麻酸(γ-linolenic acud,GLA)濃度大于 120 μM均能顯著抑制 LoVo和RKO細(xì)胞的生長,誘導(dǎo)兩細(xì)胞凋亡,但對正常細(xì)胞HUVEC影響較小,且半分化細(xì)胞RKO對PUFAs的敏感性較未分化細(xì)胞LoVo大。根據(jù)MTT結(jié)果,為了保證脂肪酸處理作用合理高效,我們選擇ALA(150μM)、EPA(150μM)、DHA(150μM)、LA(150μM)、GLA(300μM)、AA(150μM)、5-FU(100μM)處理 LoVo細(xì)胞,以 ALA(140μM)、EPA(120μM)、DHA(120 μM)、LA(120μM)、GLA(200μM)、AA(120μM)、5-FU(0.4μM)處理RKO,建立PUFAs損傷結(jié)腸癌細(xì)胞體外模型。以此濃度進(jìn)一步研究PUFAs對結(jié)腸癌細(xì)胞LoVo和RKO的線粒體損傷、脂肪酸代謝等方面的影響,發(fā)現(xiàn)存在如下效果:(1)從線粒體損傷的角度探討PUFAs對結(jié)腸癌細(xì)胞的抑制機(jī)制。結(jié)果表明,n-3(ALA、EPA、DHA)和 n-6(LA、GLA、AA)脂肪酸作用可致 LoVo 和 RKO細(xì)胞內(nèi)活性氧ROS水平和細(xì)胞漿內(nèi)Ca2+水平上升,ATP含量下降,導(dǎo)致Bcl-2家族中Bax/Bcl-2基因表達(dá)比上調(diào)。進(jìn)而引起線粒體膜出現(xiàn)小氣孔,線粒體膜電位下降,細(xì)胞色素C和AIF等蛋白釋放到胞質(zhì)中,最終激活Caspase-9和Caspase-3活性,引發(fā)Caspase級聯(lián)反應(yīng)導(dǎo)致細(xì)胞凋亡。多不飽和脂肪酸對結(jié)腸癌細(xì)胞線粒體有一定的損傷作用,通過線粒體凋亡途徑誘導(dǎo)其走向死亡。(2)研究PUFAs在LoVo和RKO細(xì)胞內(nèi)的生物轉(zhuǎn)化及其代謝產(chǎn)物對兩細(xì)胞的影響。油紅O染色實驗表明外源性添加的PUFAs通過細(xì)胞膜以脂滴形式儲存于細(xì)胞質(zhì)內(nèi)。通過氣相色譜法分析PUFAs作用下結(jié)腸癌細(xì)胞內(nèi)的脂肪酸組成,發(fā)現(xiàn)PUFAs作用后改變了 LoVo和RKO細(xì)胞內(nèi)的脂肪酸組成,提高不飽和脂肪酸比例,且在兩細(xì)胞內(nèi)n-3和n-6 PUFAs之間的轉(zhuǎn)化存在緊密聯(lián)系。此外,多不飽和脂肪酸可通過抑制結(jié)腸癌細(xì)胞COX-2、mPGES、PGDS和ALOX5的表達(dá),進(jìn)而降低脂肪酸代謝產(chǎn)物促炎因子PGE2和LTB4的分泌,促進(jìn)抗炎因子LXA4的分泌,使癌細(xì)胞趨于不利于其生長的抗炎狀態(tài)。體內(nèi)部分以C57BL/6小鼠為對象,通過分析富含長鏈脂肪酸的魚油、亞麻籽油、葵花籽油對小鼠結(jié)腸菌群結(jié)構(gòu),小鼠腸道主要代謝產(chǎn)物短鏈脂肪酸、膽汁酸,小鼠結(jié)腸組織脂類代謝參數(shù)的影響,分析多不飽和脂肪酸在改善腸道生理環(huán)境,預(yù)防結(jié)腸癌發(fā)生方面的功效。結(jié)果表明膳食長鏈脂肪酸可激活脂肪酸轉(zhuǎn)運相關(guān)基因(PPARα、OSTα)mRNA的表達(dá)促進(jìn)脂肪代謝,魚油和亞麻籽油能提高結(jié)腸組織中n-3/n-6的比值,保護(hù)結(jié)腸組織免受氧化應(yīng)激損傷。此外,膳食長鏈不飽和脂肪酸能改變小鼠腸道菌群結(jié)構(gòu),魚油和亞麻籽油處理組在Eubacterium,Novosphingobium,和Hymenobacter上的豐度高于對照組。富含n-6 PUFAs的葵花籽油組的結(jié)腸微生物多樣性不如富含n-3 PUFAs魚油組和亞麻籽油組。亞麻籽油、葵花籽油,特別是魚油中的多不飽和脂肪酸可促進(jìn)丁酸產(chǎn)生菌Eubacterium的生長,提高短鏈脂肪酸含量(特別是丁酸含量提高50%),降低腸道pH值,減少次級膽汁酸含量,起到改善腸道環(huán)境的作用,對結(jié)腸癌的發(fā)生有一定的預(yù)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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R735.35

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