高脂膳食对胰腺癌影响的实验研究

发布时间：2018-03-09 18:04

  本文选题：高脂膳食　切入点：脂肪酸分析　出处：《天津医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的:高脂膳食是胰腺癌发生发展的重要危险因素之一,本研究旨在探讨含有不同类型脂肪酸的高脂膳食对胰腺肿瘤的影响。我们以胰腺癌荷瘤小鼠为研究对象,观察分别以饱和脂肪酸、单不饱和脂肪酸、n-6和n-3多不饱和脂肪酸为主的高脂膳食对小鼠肿瘤生长的影响,比较不同类型脂肪酸对小鼠骨骼肌和肝脏组织中脂肪酸分布的影响,并初步探讨不同类型脂肪酸对胰腺癌生长影响的作用机制。方法:第一部分5周龄雄性C57BL/6裸鼠随机分为6组:饱和脂肪酸(SFA)组、单不饱和脂肪酸(MUFA)组、n-6多不饱和脂肪酸(n-6 PUFA)组、n-3多不饱和脂肪酸(n-3 PUFA)组、等能量对照(ISO-C)组和正常对照(NC)组,每组10只。四个高脂膳食组用四种油脂含量为15%(g/100g)饲料分别喂养,其油脂分别来自椰子油(富含SFA),橄榄油(富含MUFA),大豆油(富含n-6 PUFA)和亚麻籽油(富含n-3 PUFA)。ISO-C组为与四个高脂膳食组(4 kcal/g)等能量的对照组,其与NC组(3 kcal/g)的饲料均含4%～5%的大豆油。各组裸鼠在用相应的试验饲料喂养一周后,将HPAF-II胰腺癌细胞接种到胰腺内,建立胰腺癌模型,继续该饲料喂养十三周。在实验过程中观察其一般状态,记录小鼠每周的进食量及体重。实验结束时,将小鼠麻醉处死,记录肿瘤的发生情况,称重后留取肿瘤组织,同时留取股四头肌、肝脏组织备用。其中,一部分胰腺肿瘤组织用HE染色后观察其病理学情况;另一部分则进行相关免疫组织化学检测。第二部分称取0.5～1.0g骨骼肌或肝脏样品,加入2ml等比例的石油醚和苯的提取液进行研磨。待组织研磨粉碎后,移至离心管离心,取1ml上清液于试管中,加入0.5M氢氧化钠甲醇溶液2ml,摇匀后放置在30℃水浴中皂化30 min。在试管中加入2ml蒸馏水以去除水溶物,待溶液分层后取上层液用于测定。采用气相色谱-质谱联用(GM-MS)技术分析六组小鼠骨骼肌和肝脏细胞中脂肪酸的构成。第三部分将六组小鼠的胰腺肿瘤组织分别用10%福尔马林溶液固定,石蜡包埋,5μm连续切片,应用免疫组化(IHC)染色的方法分别检测胰腺肿瘤组织中中链酰基辅酶A脱氢酶(MCAD)、小窝蛋白-1(caveolin-1)、环氧化酶-2(COX-2)、缺氧诱导因子-1α(HIF-1α)、增殖细胞核抗原(PCNA)和血管内皮生长因子(VEGF)蛋白的表达情况,并应用油红O染色的方法检测胰腺肿瘤组织中脂滴聚集的情况。结果:第一部分结果显示:(1)根据每周进食量和体重变化情况,我们将实验分为四个阶段:第一阶段(1-3w)、第二阶段(4-6w)、第三阶段(7-10w)和第四阶段(11-14w)。各组小鼠的进食情况如下:第一阶段,NC组小鼠的进食量高于MUFA组和n-6PUFA组(P0.05);进入后三个阶段,NC组进食量也同时高于SFA组、n-3PUFA组和ISO-C组(P0.05)。(2)各组小鼠的体重变化如下:第一、二阶段,NC组小鼠累计体重增长大于n-6 PUFA组和n-3 PUFA组(P0.05);在第三、四阶段,NC组体重也超过了 SFA组(P0.05)。(3)n-6PUFA组小鼠总肿瘤区域和非坏死肿瘤区域大于NC组、ISO组和n-3 PUFA组(P0.05);n-3 PUFA组小鼠胰腺肿瘤出现大片坏死区域的个数最多,发生肝转移的概率最低。(4)n-3 PUFA组和ISO-C组小鼠肿瘤的重量明显低于SFA组、MUFA组和n-6 PUFA组(P0.05),且n-3 PUFA组小鼠肿瘤的重量与ISO-C和NC两个对照组比较无统计学差异。第二部分结果显示:骨骼肌和肝脏的脂肪酸构成在ISO-C组和NC组之间大致相同,以ISO-C组为基线,我们发现小鼠骨骼肌脂肪酸的变化如下:(1)属于饱和脂肪酸的十五烷酸(C15:0)、棕榈酸(C16:0)、十七烷酸(C17:0)、硬脂酸(C18:0)和花生酸(C20:0)的含量在SFA组增高(P0.05);(2)属于单不饱和脂肪酸的油酸(C18:1)的含量在MUFA组增高(P0.05);(3)属于n-6多不饱和脂肪酸的γ-亚麻酸(γC18:3)的含量在n-6 PUFA组增高(P0.05);(4)属于n-3多不饱和脂肪酸的亚麻酸(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组增高(P0.05)。小鼠肝脏脂肪酸的构成结果显示:(1)饱和脂肪酸的含量在SFA组没有增高;(2)单不饱和脂肪酸中仅二十烯酸(C20:1)的含量在MUFA组增高(P0.05);(3)n-6多不饱和脂肪酸花生四烯酸(C20:4)的含量在n-6 PUFA组增高(P0.05);(4)亚麻酸(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 组均增高(P0.05)。第三部分结果显示:(1)SFA组、MUFA组和n-6PUFA组胰腺肿瘤组织中MCAD蛋白的表达水平高于NC组、ISO-C组和n-3 PUFA组。(2)NC组和ISO-C组肿瘤组织中脂滴比较稀少,n-6PUFA组和n-3PUFA组脂滴增加,而SFA组和MUFA组的脂滴明显增多;并且,在SFA组发现有cαveolin-1的表达。(3)n-6 PUFA组和n-3 PUFA组COX-2的表达水平高于其他组。(4)MUFA组和n-6 PUFA组肿瘤组织中,HIF-1α和PCNA蛋白有共表达现象。(5)而在n-6 PUFA组肿瘤组织中,可见HIF-1α和VEGF蛋白有共表达趋势。结论:1.在胰腺癌生长过程中,荷瘤小鼠进食不同类型脂肪酸的高脂饲料及等能量的饲料并没有增加其体重及进食量;反而,正常对照组小鼠虽然进食了低能量的普通膳食,但其进食量和体重增长却高于其余各组。至实验结束时,各组小鼠均发生肿瘤。其中,n-6 PUFA组小鼠总肿瘤区域和非坏死肿瘤区域均大于NC组、ISO组和n-3 PUFA组;SFA组和MUFA组小鼠总肿瘤区域与NC组、ISO组和n-3 PUFA组相比,也呈现增大的趋势。另外在瘤重方面,n-3 PUFA组和ISO-C组小鼠肿瘤的重量明显低于SFA组、MUFA组和n-6 PUFA组。由此可推测,SFA、MUFA和n-6 PUFA能促进胰腺癌的发展,而n-3 PUFA能够抑制胰腺癌的发展。2.荷瘤小鼠骨骼肌中四类脂肪酸中的三类(SFA、MUFA和n-3 PUFA)随同种脂肪酸在膳食中的增加而增加;肝脏中仅一类脂肪酸(n-3 PUFA)随同种脂肪酸在膳食中的增加而增加。因此,骨骼肌脂肪酸的构成基本反映了饲料脂肪酸的构成;肝脏脂肪酸的构成与饲料中脂肪酸的构成部分一致。3.含不同类型脂肪酸的高脂膳食可通过不同的途径对胰腺癌细胞的生长进行调控。SFA组和MUFA组脂滴的数量显著增加,并在SFA组发现有caveolin-1的表达,SFA组、MUFA组和n-6 PUFA组MCAD蛋白的表达也增强,所以,SFA、MUFA和n-6 PUFA对胰腺癌细胞生长的促进作用可能与其增强了机体的氧化代谢有关;n-6 PUFA组和n-3 PUFA组可见COX-2表达增强,并且n-6 PUFA组可见HIF-1α与PCNA、HIF-1α与VEGF呈现共表达,所以,n-6 PUFA还可能是通过上调HIF-1α而增强胰腺癌细胞的生存能力;而n-3 PUFA可与n-6 PUFA竞争性结合COX-2,抑制PGE2的产生,并在体内转变成PGE3,从而发挥抗肿瘤的作用。本课题旨在探讨含不同类型脂肪酸的高脂膳食对胰腺癌的影响。我们通过建立荷胰腺癌小鼠模型,再分别给予小鼠含饱和脂肪酸、单不饱和脂肪酸、n-6和n-3多不饱和脂肪酸为主的高脂膳食,观察不同的脂肪酸对小鼠肿瘤生长的影响及其可能的作用机制。结果发现,骨骼肌脂肪酸的构成基本反映了饲料脂肪酸的构成;而肝脏脂肪酸的构成与饲料中脂肪酸的构成部分一致;SFA、MUFA和n-6PUFA可促进胰腺癌的发展,而n-3PUFA可抑制胰腺癌的发展。不同类型脂肪酸可通过不同的途径对胰腺癌的生长进行调控。
[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缁，

本文编号：1589645