本文選題：Caco-2細(xì)胞 + 乳糜微粒��； 參考：《華中農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：日糧脂類(lèi)中的長(zhǎng)鏈脂肪酸(Long chain fatty acid,LCFA)在進(jìn)入機(jī)體前必須在被腸上皮細(xì)胞吸收攝取后,經(jīng)歷一系列轉(zhuǎn)運(yùn)及組裝過(guò)程,最終以乳糜微粒(Chylomicron,CM)的形式分泌進(jìn)入循環(huán)系統(tǒng)。為了闡明動(dòng)物腸上皮細(xì)胞轉(zhuǎn)運(yùn)日糧中不同種類(lèi)LCFA的差異,本研究利用Caco-2細(xì)胞建立體外腸上皮細(xì)胞模型,從單層細(xì)胞膜結(jié)構(gòu)的完整性以及酶/功能性蛋白表達(dá)兩個(gè)方面評(píng)價(jià)模型的有效性;在此基礎(chǔ)上研究n-3多不飽和脂肪酸(n-3 polyunsaturated fatty acids,n-3 PUFA)對(duì)Caco-2細(xì)胞CM合成及分泌的影響,通過(guò)放射性同位素標(biāo)記技術(shù)監(jiān)測(cè)CM合成過(guò)程中兩個(gè)重要底物(甘油三酯和載脂蛋白B)的合成情況,解釋在Caco-2細(xì)胞合成及分泌CM的過(guò)程中觀(guān)察到的不同脂肪酸的差異性;考慮到肝型脂肪酸結(jié)合蛋白(Liver fatty acid binding protein,L-FABP)可以攜帶LCFA入核調(diào)控基因表達(dá),在揭示了脂肪酸處理與L-FABP表達(dá)量的關(guān)系后,構(gòu)建了L-FABP過(guò)表達(dá)載體并篩選出了有效抑制L-FABP表達(dá)的干擾序列,為后續(xù)機(jī)制研究奠定基礎(chǔ)。1.Caco-2單層細(xì)胞模型的建立。利用電阻儀測(cè)定跨上皮細(xì)胞電阻(Transepithelial electrical resistance,TEER)評(píng)價(jià)單層膜結(jié)構(gòu)的完整性和致密性,TEER值隨著培養(yǎng)時(shí)間的延長(zhǎng)呈現(xiàn)穩(wěn)定的上升趨勢(shì),分化20d時(shí)TEER值平均可以達(dá)到570Ω·cm2;功能性結(jié)構(gòu)的形成通過(guò)透射電鏡進(jìn)行確認(rèn),分化21d的Caco-2細(xì)胞具有微絨毛及緊密連接這兩個(gè)很具有代表性的腸上皮細(xì)胞特有的超微結(jié)構(gòu);堿性磷酸酶(Alkaline phosphatase,ALP)作為極性分泌的刷狀緣酶可以反應(yīng)Caco-2細(xì)胞的分化進(jìn)程,培養(yǎng)21d時(shí),ALP幾乎全部集中在頂端刷狀緣一側(cè);L-FABP作為脂質(zhì)代謝關(guān)鍵蛋白,其表達(dá)量可以反應(yīng)脂質(zhì)代謝等生理功能是否完善,隨著分化的進(jìn)行Caco-2細(xì)胞內(nèi)L-FABP表達(dá)量逐漸增加,在分化成熟的Caco-2細(xì)胞中L-FABP表達(dá)量很高。2.DHA(Docosahexaenoic acid,22:6)及EPA(eicosapentaenoic acid,20:5)對(duì)Caco-2細(xì)胞CM組裝分泌的影響。MTT試驗(yàn)分析脂肪酸濃度對(duì)Caco-2細(xì)胞的脂質(zhì)毒性發(fā)現(xiàn),600μmol/L濃度OA(Oleic acid,18:1)處理下細(xì)胞的存活力仍然可以達(dá)到95%;往上室培養(yǎng)基中加入[1,2,3-3H]glycerol研究TG合成情況,相比對(duì)照組,400μmol/L濃度的脂肪酸處理均可以顯著增加胞內(nèi)聚集的及培養(yǎng)基中分泌的[3H]triglyceride的量(P0.01),標(biāo)記的TG的量隨著孵育時(shí)間的延長(zhǎng)而增加,直到36h時(shí),脂肪酸對(duì)TG合成的差異性才顯現(xiàn)出來(lái),相比OA處理組,EPA處理組細(xì)胞內(nèi)聚集的以及培養(yǎng)基中分泌的[3H]triglyceride的量分別降低了16.5%和21%,DHA處理組則分別降低了23.2%和28%(P0.01);向上室培養(yǎng)基中加入[1-14C]脂肪酸檢測(cè)細(xì)胞對(duì)不同脂肪酸的攝取效率,上室培養(yǎng)基中放射性元素的消失率及攝取進(jìn)入細(xì)胞的放射性元素的強(qiáng)度都相同(P0.05),在處理12h~24h間約有50%~80%的脂肪酸進(jìn)入細(xì)胞內(nèi),下室培養(yǎng)基中放射性元素的強(qiáng)度在三種脂肪酸處理間也沒(méi)有差異(P0.05);向上室培養(yǎng)基中加入[35S]methionine檢測(cè)載脂蛋白B(Apolipoprotein B,apo B)的合成情況,三種脂肪酸處理下,不管是胞內(nèi)聚集的還是培養(yǎng)基中分泌的被標(biāo)記的apo B48及apo B100的量都顯著增加(P0.01),EPA和DHA處理組顯著低于OA處理組(P0.01),EPA和DHA處理組之間差異不顯著(P0.05);利用密度梯度離心分離測(cè)定脂蛋白發(fā)現(xiàn),相比相同濃度的OA,400μmol/L的EPA和DHA可以顯著降低Caco-2細(xì)胞CM的分泌量(P0.01)。3.脂肪酸處理與L-FABP表達(dá)量之間關(guān)系的確定及L-FABP過(guò)表達(dá)/干擾質(zhì)粒的構(gòu)建篩選。400μmol/L的OA、EPA和DHA刺激36h均顯著增加了Caco-2細(xì)胞內(nèi)L-FABP的表達(dá)量(P0.01),三種脂肪酸在刺激L-FABP表達(dá)的效果上沒(méi)有差異(P0.05);設(shè)計(jì)的4條sh RNA干擾載體分別與測(cè)序正確的過(guò)表達(dá)載體共轉(zhuǎn)染293T細(xì)胞,利用western檢測(cè)L-FABP的表達(dá),驗(yàn)證了過(guò)表達(dá)質(zhì)�？梢杂行Хg形成L-FABP,設(shè)計(jì)的干擾序列中sh RNA2載體的干擾效率最高;轉(zhuǎn)染Caco-2細(xì)胞,干擾組L-FABP的表達(dá)量顯著低于干擾對(duì)照組(P0.01),過(guò)表達(dá)組L-FABP的表達(dá)量顯著高于過(guò)表達(dá)對(duì)照組(P0.01)。為后續(xù)探究脂肪酸對(duì)CM組裝分泌的影響是否依賴(lài)于L-FABP/PPARα信號(hào)通路奠定基礎(chǔ)。以上結(jié)果表明:本試驗(yàn)條件下極性分化完全的Caco-2細(xì)胞可以作為研究小腸營(yíng)養(yǎng)物質(zhì)吸收代謝的體外模型,用于后續(xù)脂質(zhì)代謝的相關(guān)試驗(yàn);相比OA,EPA和DHA抑制Caco-2細(xì)胞CM分泌的作用并非由于脂肪酸攝取的差異造成,而是受TG及apo B合成減少的影響。闡明了日糧中不同LCFA對(duì)動(dòng)物腸上皮細(xì)胞內(nèi)CM組裝分泌的影響及分子機(jī)制,完善動(dòng)物腸上皮細(xì)胞轉(zhuǎn)運(yùn)日糧脂肪的基礎(chǔ)理論。
[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.

【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：S816

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