EPA和DHA调控Caco-2细胞乳糜微粒组装的机制研究

发布时间：2018-05-01 00:09

本文选题：Caco-2细胞 + 乳糜微粒　；参考：《华中农业大学》2015年硕士论文

【摘要】：日粮脂类中的长链脂肪酸(Long chain fatty acid,LCFA)在进入机体前必须在被肠上皮细胞吸收摄取后,经历一系列转运及组装过程,最终以乳糜微粒(Chylomicron,CM)的形式分泌进入循环系统。为了阐明动物肠上皮细胞转运日粮中不同种类LCFA的差异,本研究利用Caco-2细胞建立体外肠上皮细胞模型,从单层细胞膜结构的完整性以及酶/功能性蛋白表达两个方面评价模型的有效性;在此基础上研究n-3多不饱和脂肪酸(n-3 polyunsaturated fatty acids,n-3 PUFA)对Caco-2细胞CM合成及分泌的影响,通过放射性同位素标记技术监测CM合成过程中两个重要底物(甘油三酯和载脂蛋白B)的合成情况,解释在Caco-2细胞合成及分泌CM的过程中观察到的不同脂肪酸的差异性;考虑到肝型脂肪酸结合蛋白(Liver fatty acid binding protein,L-FABP)可以携带LCFA入核调控基因表达,在揭示了脂肪酸处理与L-FABP表达量的关系后,构建了L-FABP过表达载体并筛选出了有效抑制L-FABP表达的干扰序列,为后续机制研究奠定基础。1.Caco-2单层细胞模型的建立。利用电阻仪测定跨上皮细胞电阻(Transepithelial electrical resistance,TEER)评价单层膜结构的完整性和致密性,TEER值随着培养时间的延长呈现稳定的上升趋势,分化20d时TEER值平均可以达到570Ω·cm2;功能性结构的形成通过透射电镜进行确认,分化21d的Caco-2细胞具有微绒毛及紧密连接这两个很具有代表性的肠上皮细胞特有的超微结构;碱性磷酸酶(Alkaline phosphatase,ALP)作为极性分泌的刷状缘酶可以反应Caco-2细胞的分化进程,培养21d时,ALP几乎全部集中在顶端刷状缘一侧;L-FABP作为脂质代谢关键蛋白,其表达量可以反应脂质代谢等生理功能是否完善,随着分化的进行Caco-2细胞内L-FABP表达量逐渐增加,在分化成熟的Caco-2细胞中L-FABP表达量很高。2.DHA(Docosahexaenoic acid,22:6)及EPA(eicosapentaenoic acid,20:5)对Caco-2细胞CM组装分泌的影响。MTT试验分析脂肪酸浓度对Caco-2细胞的脂质毒性发现,600μmol/L浓度OA(Oleic acid,18:1)处理下细胞的存活力仍然可以达到95%;往上室培养基中加入[1,2,3-3H]glycerol研究TG合成情况,相比对照组,400μmol/L浓度的脂肪酸处理均可以显著增加胞内聚集的及培养基中分泌的[3H]triglyceride的量(P0.01),标记的TG的量随着孵育时间的延长而增加,直到36h时,脂肪酸对TG合成的差异性才显现出来,相比OA处理组,EPA处理组细胞内聚集的以及培养基中分泌的[3H]triglyceride的量分别降低了16.5%和21%,DHA处理组则分别降低了23.2%和28%(P0.01);向上室培养基中加入[1-14C]脂肪酸检测细胞对不同脂肪酸的摄取效率,上室培养基中放射性元素的消失率及摄取进入细胞的放射性元素的强度都相同(P0.05),在处理12h~24h间约有50%~80%的脂肪酸进入细胞内,下室培养基中放射性元素的强度在三种脂肪酸处理间也没有差异(P0.05);向上室培养基中加入[35S]methionine检测载脂蛋白B(Apolipoprotein B,apo B)的合成情况,三种脂肪酸处理下,不管是胞内聚集的还是培养基中分泌的被标记的apo B48及apo B100的量都显著增加(P0.01),EPA和DHA处理组显著低于OA处理组(P0.01),EPA和DHA处理组之间差异不显著(P0.05);利用密度梯度离心分离测定脂蛋白发现,相比相同浓度的OA,400μmol/L的EPA和DHA可以显著降低Caco-2细胞CM的分泌量(P0.01)。3.脂肪酸处理与L-FABP表达量之间关系的确定及L-FABP过表达/干扰质粒的构建筛选。400μmol/L的OA、EPA和DHA刺激36h均显著增加了Caco-2细胞内L-FABP的表达量(P0.01),三种脂肪酸在刺激L-FABP表达的效果上没有差异(P0.05);设计的4条sh RNA干扰载体分别与测序正确的过表达载体共转染293T细胞,利用western检测L-FABP的表达,验证了过表达质粒可以有效翻译形成L-FABP,设计的干扰序列中sh RNA2载体的干扰效率最高;转染Caco-2细胞,干扰组L-FABP的表达量显著低于干扰对照组(P0.01),过表达组L-FABP的表达量显著高于过表达对照组(P0.01)。为后续探究脂肪酸对CM组装分泌的影响是否依赖于L-FABP/PPARα信号通路奠定基础。以上结果表明:本试验条件下极性分化完全的Caco-2细胞可以作为研究小肠营养物质吸收代谢的体外模型,用于后续脂质代谢的相关试验;相比OA,EPA和DHA抑制Caco-2细胞CM分泌的作用并非由于脂肪酸摄取的差异造成,而是受TG及apo B合成减少的影响。阐明了日粮中不同LCFA对动物肠上皮细胞内CM组装分泌的影响及分子机制,完善动物肠上皮细胞转运日粮脂肪的基础理论。
[Abstract]:Long chain fatty acid (LCFA) in dietary lipids must undergo a series of transport and assembly processes before being absorbed into the intestinal epithelial cells and eventually secreted into the circulatory system in the form of Chylomicron (CM). In order to elucidate the different types of LCFA in the animal intestinal epithelial cells. In this study, Caco-2 cells were used to build a stereoscopic outer intestinal epithelial cell model and evaluate the validity of the model from two aspects of the integrity of the monolayer membrane structure and the expression of the enzyme / functional protein expression. On this basis, the effects of n-3 polyunsaturated fatty acids (n-3 polyunsaturated fatty acids, n-3 PUFA) on the synthesis and secretion of Caco-2 cell CM were studied. To monitor the synthesis of two important substrates (triglycerides and apolipoprotein B) during CM synthesis by radioisotope labeling technique, explaining the difference in the difference in fatty acids observed during the synthesis and secretion of CM in Caco-2 cells, and considering the liver type fatty acid junction protein (Liver fatty acid binding protein, L-FABP) In order to regulate the expression of LCFA gene, after revealing the relationship between fatty acid treatment and the expression of L-FABP, the L-FABP overexpression vector was constructed and the interference sequence to effectively inhibit the expression of L-FABP was screened, and the foundation of the basic.1.Caco-2 cell model for the follow-up mechanism was established. The resistance (Trans) was determined by the resistance meter (Trans). Epithelial electrical resistance, TEER) evaluated the integrity and densification of the monolayer structure. TEER value increased steadily with the prolongation of the incubation time. The average TEER value of the differentiated 20d could reach 570 Omega cm2; the formation of functional structure was identified by transmission electron microscopy, and the Caco-2 cells of the differentiated 21d were microvilli and tight. Connecting these two very representative intestinal epithelial cells specific ultrastructure; alkaline phosphatase (Alkaline phosphatase, ALP) as a polar secreted brush margin enzyme can react to the differentiation process of Caco-2 cells. When 21d, ALP is almost all concentrated on the tip of the top edge of the top of the brush; L-FABP is the key protein of lipid metabolism, and its expression can be expressed. With the improvement of lipid metabolism and other physiological functions, the expression of L-FABP in the differentiated Caco-2 cells increased gradually with the differentiation of Caco-2 cells. The effect of.2.DHA (Docosahexaenoic acid, 22:6) and EPA (eicosapentaenoic acid, 20:5) on the secretion of fatty acids in the differentiated Caco-2 cells The lipid toxicity of concentration on Caco-2 cells showed that the viability of cells under the concentration of OA (Oleic acid, 18:1) was still up to 95%. [1,2,3-3H]glycerol was added to the upper chamber culture medium to study the synthesis of TG. Compared with the control group, the fatty acids at 400 mu concentration could significantly increase the intracellular aggregation and medium fraction. The amount of [3H]triglyceride (P0.01), the amount of labeled TG increased with the prolongation of incubation time. The difference of fatty acids to TG synthesis was revealed until 36h. Compared to the OA treatment group, the intracellular aggregation of EPA treatment group and the amount of [3H] triglyceride secreted in the medium decreased by 16.5% and 21% respectively, and the DHA treatment group decreased respectively. 23.2% and 28% (P0.01) were lower; [1-14C] fatty acids were added into the upper chamber medium to detect the uptake efficiency of cells to different fatty acids. The disappearance of radioactive elements in the room culture medium and the intensity of the radioactive elements taken into the cells were all the same (P0.05). The fatty acids, which were 50%~80% in the treatment of 12h~24h, entered the cell and the cell culture medium. There was no difference in the intensity of the radioactive elements between the three fatty acids (P0.05); the synthesis of apolipoprotein B (Apolipoprotein B, apo B) was detected by [35S]methionine in the upper chamber culture medium, and the amount of the labeled apo B48 and apo B100 secreted in the three fatty acids, both in the cell and in the medium, was significant. The increase (P0.01), EPA and DHA treatment group were significantly lower than that in OA treatment group (P0.01), and there was no significant difference between EPA and DHA treatment groups (P0.05). The determination of lipoprotein by density gradient centrifugation was compared with the same concentration of OA. The EPA and DHA of 400 mu mol/L could significantly reduce the secretion of fatty acid and the expression of fatty acids. The determination of the inter relationship and the construction of L-FABP overexpression / interference plasmids to screen the OA of.400 mu mol/L, EPA and DHA stimulation 36h significantly increased the L-FABP expression in Caco-2 cells (P0.01), and there was no difference in the effect of three kinds of fatty acids on the L-FABP expression (P0.05); the 4 designed interference carriers were in common with the correct overexpression vector of sequencing, respectively. The transfection of 293T cells and using western to detect the expression of L-FABP showed that the overexpressed plasmid could effectively translate and form L-FABP. The interference efficiency of SH RNA2 vector was the highest in the designed interference sequence, and the expression of L-FABP in the transfected Caco-2 cells was significantly lower than that of the interference control group (P0.01), and the expression of L-FABP in the overexpressed group was significantly higher than that of overexpression. The control group (P0.01). To investigate whether the effect of fatty acids on CM assembly and secretion depends on the L-FABP/PPAR alpha signaling pathway. The above results suggest that Caco-2 cells with complete polarity differentiation under this experimental condition can be used as an in vitro model for the study of absorption and metabolism of small intestinal nutrients and for subsequent lipid metabolism related experiments; The inhibitory effect of OA, EPA and DHA on the secretion of CM in Caco-2 cells was not caused by the difference in fatty acid uptake, but was influenced by the decrease in the synthesis of TG and apo B. The effects of different LCFA on the secretion of CM assembly and secretion of CM in the intestinal epithelial cells of the animals were elucidated, and the basic theory of the transport of fat in the animal intestinal epithelial cells was improved.

【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S816

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