本文選題：高脂膳食　切入點：脂肪酸分析　出處：《天津醫(yī)科大學》2014年碩士論文　論文類型：學位論文

【摘要】：目的:高脂膳食是胰腺癌發(fā)生發(fā)展的重要危險因素之一,本研究旨在探討含有不同類型脂肪酸的高脂膳食對胰腺腫瘤的影響。我們以胰腺癌荷瘤小鼠為研究對象,觀察分別以飽和脂肪酸、單不飽和脂肪酸、n-6和n-3多不飽和脂肪酸為主的高脂膳食對小鼠腫瘤生長的影響,比較不同類型脂肪酸對小鼠骨骼肌和肝臟組織中脂肪酸分布的影響,并初步探討不同類型脂肪酸對胰腺癌生長影響的作用機制。方法:第一部分5周齡雄性C57BL/6裸鼠隨機分為6組:飽和脂肪酸(SFA)組、單不飽和脂肪酸(MUFA)組、n-6多不飽和脂肪酸(n-6 PUFA)組、n-3多不飽和脂肪酸(n-3 PUFA)組、等能量對照(ISO-C)組和正常對照(NC)組,每組10只。四個高脂膳食組用四種油脂含量為15%(g/100g)飼料分別喂養(yǎng),其油脂分別來自椰子油(富含SFA),橄欖油(富含MUFA),大豆油(富含n-6 PUFA)和亞麻籽油(富含n-3 PUFA)。ISO-C組為與四個高脂膳食組(4 kcal/g)等能量的對照組,其與NC組(3 kcal/g)的飼料均含4%～5%的大豆油。各組裸鼠在用相應(yīng)的試驗飼料喂養(yǎng)一周后,將HPAF-II胰腺癌細胞接種到胰腺內(nèi),建立胰腺癌模型,繼續(xù)該飼料喂養(yǎng)十三周。在實驗過程中觀察其一般狀態(tài),記錄小鼠每周的進食量及體重。實驗結(jié)束時,將小鼠麻醉處死,記錄腫瘤的發(fā)生情況,稱重后留取腫瘤組織,同時留取股四頭肌、肝臟組織備用。其中,一部分胰腺腫瘤組織用HE染色后觀察其病理學情況;另一部分則進行相關(guān)免疫組織化學檢測。第二部分稱取0.5～1.0g骨骼肌或肝臟樣品,加入2ml等比例的石油醚和苯的提取液進行研磨。待組織研磨粉碎后,移至離心管離心,取1ml上清液于試管中,加入0.5M氫氧化鈉甲醇溶液2ml,搖勻后放置在30℃水浴中皂化30 min。在試管中加入2ml蒸餾水以去除水溶物,待溶液分層后取上層液用于測定。采用氣相色譜-質(zhì)譜聯(lián)用(GM-MS)技術(shù)分析六組小鼠骨骼肌和肝臟細胞中脂肪酸的構(gòu)成。第三部分將六組小鼠的胰腺腫瘤組織分別用10%福爾馬林溶液固定,石蠟包埋,5μm連續(xù)切片,應(yīng)用免疫組化(IHC)染色的方法分別檢測胰腺腫瘤組織中中鏈酰基輔酶A脫氫酶(MCAD)、小窩蛋白-1(caveolin-1)、環(huán)氧化酶-2(COX-2)、缺氧誘導因子-1α(HIF-1α)、增殖細胞核抗原(PCNA)和血管內(nèi)皮生長因子(VEGF)蛋白的表達情況,并應(yīng)用油紅O染色的方法檢測胰腺腫瘤組織中脂滴聚集的情況。結(jié)果:第一部分結(jié)果顯示:(1)根據(jù)每周進食量和體重變化情況,我們將實驗分為四個階段:第一階段(1-3w)、第二階段(4-6w)、第三階段(7-10w)和第四階段(11-14w)。各組小鼠的進食情況如下:第一階段,NC組小鼠的進食量高于MUFA組和n-6PUFA組(P0.05);進入后三個階段,NC組進食量也同時高于SFA組、n-3PUFA組和ISO-C組(P0.05)。(2)各組小鼠的體重變化如下:第一、二階段,NC組小鼠累計體重增長大于n-6 PUFA組和n-3 PUFA組(P0.05);在第三、四階段,NC組體重也超過了 SFA組(P0.05)。(3)n-6PUFA組小鼠總腫瘤區(qū)域和非壞死腫瘤區(qū)域大于NC組、ISO組和n-3 PUFA組(P0.05);n-3 PUFA組小鼠胰腺腫瘤出現(xiàn)大片壞死區(qū)域的個數(shù)最多,發(fā)生肝轉(zhuǎn)移的概率最低。(4)n-3 PUFA組和ISO-C組小鼠腫瘤的重量明顯低于SFA組、MUFA組和n-6 PUFA組(P0.05),且n-3 PUFA組小鼠腫瘤的重量與ISO-C和NC兩個對照組比較無統(tǒng)計學差異。第二部分結(jié)果顯示:骨骼肌和肝臟的脂肪酸構(gòu)成在ISO-C組和NC組之間大致相同,以ISO-C組為基線,我們發(fā)現(xiàn)小鼠骨骼肌脂肪酸的變化如下:(1)屬于飽和脂肪酸的十五烷酸(C15:0)、棕櫚酸(C16:0)、十七烷酸(C17:0)、硬脂酸(C18:0)和花生酸(C20:0)的含量在SFA組增高(P0.05);(2)屬于單不飽和脂肪酸的油酸(C18:1)的含量在MUFA組增高(P0.05);(3)屬于n-6多不飽和脂肪酸的γ-亞麻酸(γC18:3)的含量在n-6 PUFA組增高(P0.05);(4)屬于n-3多不飽和脂肪酸的亞麻酸(C18:3)、二十碳五烯酸(C20:5)和二十二碳五烯酸(C22:5)的含量在n-3 PUFA組增高(P0.05)。小鼠肝臟脂肪酸的構(gòu)成結(jié)果顯示:(1)飽和脂肪酸的含量在SFA組沒有增高;(2)單不飽和脂肪酸中僅二十烯酸(C20:1)的含量在MUFA組增高(P0.05);(3)n-6多不飽和脂肪酸花生四烯酸(C20:4)的含量在n-6 PUFA組增高(P0.05);(4)亞麻酸(C18:3)、二十碳五烯酸(C20:5)、二十二碳五烯酸(C22:5)的含量在n-3 PUFA 組均增高(P0.05)。第三部分結(jié)果顯示:(1)SFA組、MUFA組和n-6PUFA組胰腺腫瘤組織中MCAD蛋白的表達水平高于NC組、ISO-C組和n-3 PUFA組。(2)NC組和ISO-C組腫瘤組織中脂滴比較稀少,n-6PUFA組和n-3PUFA組脂滴增加,而SFA組和MUFA組的脂滴明顯增多;并且,在SFA組發(fā)現(xiàn)有cαveolin-1的表達。(3)n-6 PUFA組和n-3 PUFA組COX-2的表達水平高于其他組。(4)MUFA組和n-6 PUFA組腫瘤組織中,HIF-1α和PCNA蛋白有共表達現(xiàn)象。(5)而在n-6 PUFA組腫瘤組織中,可見HIF-1α和VEGF蛋白有共表達趨勢。結(jié)論:1.在胰腺癌生長過程中,荷瘤小鼠進食不同類型脂肪酸的高脂飼料及等能量的飼料并沒有增加其體重及進食量;反而,正常對照組小鼠雖然進食了低能量的普通膳食,但其進食量和體重增長卻高于其余各組。至實驗結(jié)束時,各組小鼠均發(fā)生腫瘤。其中,n-6 PUFA組小鼠總腫瘤區(qū)域和非壞死腫瘤區(qū)域均大于NC組、ISO組和n-3 PUFA組;SFA組和MUFA組小鼠總腫瘤區(qū)域與NC組、ISO組和n-3 PUFA組相比,也呈現(xiàn)增大的趨勢。另外在瘤重方面,n-3 PUFA組和ISO-C組小鼠腫瘤的重量明顯低于SFA組、MUFA組和n-6 PUFA組。由此可推測,SFA、MUFA和n-6 PUFA能促進胰腺癌的發(fā)展,而n-3 PUFA能夠抑制胰腺癌的發(fā)展。2.荷瘤小鼠骨骼肌中四類脂肪酸中的三類(SFA、MUFA和n-3 PUFA)隨同種脂肪酸在膳食中的增加而增加;肝臟中僅一類脂肪酸(n-3 PUFA)隨同種脂肪酸在膳食中的增加而增加。因此,骨骼肌脂肪酸的構(gòu)成基本反映了飼料脂肪酸的構(gòu)成;肝臟脂肪酸的構(gòu)成與飼料中脂肪酸的構(gòu)成部分一致。3.含不同類型脂肪酸的高脂膳食可通過不同的途徑對胰腺癌細胞的生長進行調(diào)控。SFA組和MUFA組脂滴的數(shù)量顯著增加,并在SFA組發(fā)現(xiàn)有caveolin-1的表達,SFA組、MUFA組和n-6 PUFA組MCAD蛋白的表達也增強,所以,SFA、MUFA和n-6 PUFA對胰腺癌細胞生長的促進作用可能與其增強了機體的氧化代謝有關(guān);n-6 PUFA組和n-3 PUFA組可見COX-2表達增強,并且n-6 PUFA組可見HIF-1α與PCNA、HIF-1α與VEGF呈現(xiàn)共表達,所以,n-6 PUFA還可能是通過上調(diào)HIF-1α而增強胰腺癌細胞的生存能力;而n-3 PUFA可與n-6 PUFA競爭性結(jié)合COX-2,抑制PGE2的產(chǎn)生,并在體內(nèi)轉(zhuǎn)變成PGE3,從而發(fā)揮抗腫瘤的作用。本課題旨在探討含不同類型脂肪酸的高脂膳食對胰腺癌的影響。我們通過建立荷胰腺癌小鼠模型,再分別給予小鼠含飽和脂肪酸、單不飽和脂肪酸、n-6和n-3多不飽和脂肪酸為主的高脂膳食,觀察不同的脂肪酸對小鼠腫瘤生長的影響及其可能的作用機制。結(jié)果發(fā)現(xiàn),骨骼肌脂肪酸的構(gòu)成基本反映了飼料脂肪酸的構(gòu)成;而肝臟脂肪酸的構(gòu)成與飼料中脂肪酸的構(gòu)成部分一致;SFA、MUFA和n-6PUFA可促進胰腺癌的發(fā)展,而n-3PUFA可抑制胰腺癌的發(fā)展。不同類型脂肪酸可通過不同的途徑對胰腺癌的生長進行調(diào)控。
[Abstract]:Purpose: high fat diet is one of the important risk factors for the occurrence and development of pancreatic cancer, this study aimed to investigate the effect of high fat diet containing different types of fatty acids on pancreatic cancer. We use mouse pancreatic cancer tumor bearing as the research object, observe respectively with saturated fatty acids, monounsaturated fatty acids and n-6, and n-3 effect of unsaturated fatty acid of high fat diet based on tumor growth in mice, comparison of different types of fatty acids on the fatty acid distribution of mouse skeletal muscle and liver tissue, and to investigate the effects of different types of fatty acids on the growth of pancreatic cancer molecular mechanisms. Methods: in the first part of the 5 week old male C57BL/6 mice were randomly divided into into 6 groups: saturated fatty acid (SFA) group, monounsaturated fatty acid (MUFA) group, n-6 polyunsaturated fatty acids (n-6 PUFA) group, n-3 polyunsaturated fatty acids (n-3 PUFA) group, energy control (ISO-C) group and normal control group (NC), each Group 10. Four high fat diet group with four kinds of oil content was 15% (g/100g) respectively, feed feeding, the oil from coconut oil (rich in SFA), olive oil (rich in MUFA), soybean oil (rich in n-6 PUFA) and linseed oil (rich in n-3 PUFA) in.ISO-C group and four high fat diet group (4 kcal/g) control group and energy, with the NC group (3 kcal/g) soybean oil 5% forage containing 4% ~. The nude mice in each group fed with the corresponding test after a week, HPAF-II pancreatic cancer cells were inoculated into the pancreas, establish a model of pancreatic cancer, to the feed keep thirteen weeks. To observe the general condition during the experiment, mice were recorded weekly food intake and body weight. At the end of the experiment, the mice were killed, the tumor occurrence records, after weighing specimens from the tumor tissue, and take stock four muscles, liver reserve. Among them, a part of pancreatic cancer tissue HE staining after view Study the pathological observation; the other part is to detect chemical related immune organization. In the second part, weighing 0.5 ~ 1.0g of skeletal muscle or liver samples, extract into 2ml ratio of petroleum ether and benzene by grinding. The grinding organization, to centrifuge tube centrifugation, 1ml supernatant liquid in a test tube, adding 0.5M 2ml sodium hydroxide methanol solution, shake after saponification placed in 30 DEG C water bath for 30 min. in a test tube, add 2ml distilled water to remove water soluble, until the solution after stratification from the upper liquid for determination. By using gas chromatography-mass spectrometry (GM-MS) technology to analyze the fatty acid composition of the six groups of mice skeletal muscle liver cells. The third part of the six groups of mice pancreatic tumor tissues were 10% formalin fixed, paraffin embedded, 5 m serial sections, immunohistochemical (IHC) staining method were used to detect pancreatic tumor in Kiev - chain The enzyme A dehydrogenase (MCAD), -1 (caveolin-1), caveolin cyclooxygenase -2 (COX-2), hypoxia inducible factor -1 alpha (HIF-1 alpha), proliferating cell nuclear antigen (PCNA) and vascular endothelial growth factor (VEGF) expression of protein, lipid droplet accumulation was detected in pancreatic tumor tissue and application methods of oil red O staining. Results: in the first part, results showed that: (1) according to the weekly food intake and body weight changes, we will experiment is divided into four stages: the first stage (1-3W), the second stage (4-6w), third (7-10w) and the fourth stage (11-14w). The mice ate as follows: the first stage, the food intake of mice in NC group was higher than that of MUFA group and n-6PUFA group (P0.05); the three stage, group NC intake also higher than that in SFA group, n-3PUFA group and ISO-C group (P0.05). (2) the mice weight changes are as follows: first, second, the cumulative weight gain of mice in the NC group more than n-6 PUFA group 鍜宯-3 PUFA緇，

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