發(fā)布時間：2017-12-28 09:41

  本文關(guān)鍵詞：谷氨酰胺通過抑制microRNA-29a和細胞自噬調(diào)控宮內(nèi)生長受限仔豬腸道發(fā)育的研究　出處：《中國農(nóng)業(yè)大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 宮內(nèi)生長受限 腸道發(fā)育 谷氨酰胺 MicroRNA-29a 自噬 營養(yǎng) 豬

【摘要】：腸道是營養(yǎng)物質(zhì)消化吸收的主要場所。宮內(nèi)生長受限(Intrauterine growth restriction,IUGR)可導(dǎo)致仔豬小腸絨毛萎縮,腸道指數(shù)下降,細胞骨架蛋白所占比例增加,提示IUGR仔豬腸道細胞增殖與發(fā)育受阻。谷氨酰胺(L-Glutamine,Gln)是腸細胞的主要能量來源,在維持正常的腸道屏障功能、調(diào)節(jié)細胞蛋白質(zhì)周轉(zhuǎn)以及促進受損腸道的修復(fù)中發(fā)揮重要作用。且相對于正常胎豬,IUGR胎豬臍帶血和羊水中Gln含量較低,推測IUGR胎豬的Gln供給不足是IUGR新生仔豬腸道發(fā)育受損的重要原因之一。本研究通過四個試驗探討IUGR新生仔豬腸道中微小RNA(MicroRNA,miRNA)對細胞增殖的調(diào)控以及Gln影響細胞自噬的發(fā)生,并研究母體添加Gln對新生仔豬生長和腸道發(fā)育的影響,分別從分子生物學(xué)角度和營養(yǎng)學(xué)角度揭示了 IUGR豬腸道發(fā)育損傷的機制和Gln改善仔豬腸道發(fā)育的機理。試驗一通過miRNA芯片篩選出miR-29家族在IUGR仔豬空腸中顯著高表達(P0.05),初步生物信息學(xué)預(yù)測miR-29a的靶基因主要集中在細胞外基質(zhì)與黏附連接信號通路。Western blot分析發(fā)現(xiàn)IUGR仔豬腸道中細胞外基質(zhì)(Fibronectin、Collagen和Integrin β1)以及緊密連接蛋白(Claudin-1、Occludin和ZO-1)表達量顯著低于正常仔豬(P0.05),驗證了 IUGR損害腸道胞外基質(zhì)-整合素信號通路。以新生仔豬空腸上皮細胞系(Porcine intestinal epithelial cell line-1,IPEC-1)為體外模型,證實了 miR-29a對細胞外基質(zhì)、細胞黏附和緊密連接基因與蛋白的靶向抑制效果,從而抑制腸道細胞增殖與整合性。本研究提示miR-29a可作為腸道黏膜細胞發(fā)育與損傷程度的一項重要檢測靶標和干預(yù)靶標。試驗二旨在探討腸道中Gln含量變化及自噬水平對IUGR胎豬腸道細胞功能的影響。取妊娠d 110的胎豬空腸粘膜樣品,高效液相色譜法檢測其游離氨基酸譜并Western blot分析正常和IUGR胎豬腸道自噬標志分子微管相關(guān)蛋白輕鏈 3B(Microtubule-associated protein 1 light chain 3B,LC3B)的表達變化。結(jié)果表明:與正常胎豬相比,IUGR胎豬腸道中Gln含量下降了 66%(P0.01),而自噬標志分子LC3B-ⅡI的表達水平則為正常仔豬腸道中的2倍(P0.05)。體外IPEC-1細胞模型研究發(fā)現(xiàn)Gln缺乏可顯著抑制IPEC-1細胞的增殖,透射電鏡觀察發(fā)現(xiàn)在0～8 h內(nèi)隨著處理時間的延長細胞內(nèi)自噬泡數(shù)量逐步增加(P0.05),自噬標志分子LC3B-Ⅱ的表達量也呈現(xiàn)相同趨勢(P0.05)。同時,Gln缺乏改變了 mTOR及MAPK信號通路相關(guān)蛋白的表達水平。Gln重補充能緩解上述現(xiàn)象發(fā)生。試驗三旨在探討母體Gln添加對新生仔豬生長和腸道發(fā)育的影響。通過在妊娠期最后30天的母豬飼糧中添加1%的Gln可增加仔豬窩平均個體重(P0.05),降低窩內(nèi)變異系數(shù)(P0.05)。利用高效液相色譜和生化分析技術(shù)分析了母豬和新生仔豬血漿游離氨基酸譜和血液生化指標,探討母體對Gln添加的自身營養(yǎng)狀況響應(yīng)及母胎營養(yǎng)物質(zhì)轉(zhuǎn)運變化規(guī)律。仔豬屠宰分析發(fā)現(xiàn)妊娠后期母體添加Gln提高了仔豬相對腸道重量(P0.05),增加了仔豬空腸絨毛高度和寬度、絨毛高度與隱窩深度比、黏膜厚度以及絨毛表面積(P0.05)。試驗四在試驗三的基礎(chǔ)上,發(fā)現(xiàn)母體Gln添加顯著抑制仔豬空腸組織中miR-29a的表達水平(P0.05),促進胞外基質(zhì)和緊密連接蛋白表達(P0.05);同時提高mTOR信號通路mTOR及其下游蛋白p70S6K的磷酸化水平(P0.05),提示Gln可促進腸道m(xù)TOR信號通路與增強細胞間緊密連接,進而達到維護腸道健康和促進腸道發(fā)育的作用。結(jié)合1PEC-1細胞模型,探討添加不同濃度的Gln對仔豬腸道細胞增殖及相關(guān)基因和蛋白表達情況的影響。結(jié)果發(fā)現(xiàn):添加Gln可促進腸道上皮細胞增殖并且提高單層細胞整合性(P0.05);同時抑制miRNA-29a的表達(P0.05),提高了細胞中胞外基質(zhì)和緊密連接蛋白等相關(guān)的基因和蛋白表達水平(P0.05),改變了相關(guān)轉(zhuǎn)錄因子的mRNA表達水平,提示Gln有可能通過抑制miR-29a表達保護細胞外基質(zhì)信號通路,進而達到維護腸道健康、促進腸道發(fā)育的作用。綜上所述,本研究結(jié)果表明在IUGR仔豬腸道中miR-29a的高表達靶向抑制細胞外基質(zhì)-整合素信號通路,Gln缺乏誘導(dǎo)細胞自噬發(fā)生并改變mTOR及MAPK信號通路表達;在妊娠后期母豬飼糧中添加Gln促進了仔豬生長和腸道發(fā)育以及相關(guān)蛋白表達,闡明了 IUGR仔豬腸道發(fā)育損傷的分子機制以及Gln在豬腸道中的保護作用,為進一步優(yōu)化母體營養(yǎng)方案促進仔豬生長提供了新的理論依據(jù)。
[Abstract]:The intestinal tract is the main place for the digestion and absorption of nutrients. Intrauterine growth restriction (Intrauterine growth, restriction, IUGR) can lead to small intestinal villi atrophy, intestinal index decreased, the increase in the proportion of cytoskeletal proteins, suggesting that IUGR piglets tract cell proliferation and impaired development. L-Glutamine (Gln) is the main energy source of intestinal cells. It plays an important role in maintaining normal intestinal barrier function, regulating cell protein turnover and promoting the repair of damaged intestinal tract. Compared with normal fetal pigs, the Gln content in IUGR fetuses, umbilical cord blood and amniotic fluid is relatively low. It is speculated that insufficient Gln supply in IUGR fetal pigs is one of the important reasons for the intestinal development of IUGR newborn piglets. Four experiments to investigate the tiny RNA IUGR in newborn piglets intestine (MicroRNA, miRNA) on cell proliferation regulation and the effect of Gln on autophagy, and study the maternal effect of Gln addition on growth and intestinal development of newborn piglets, respectively, from the perspective of molecular biology and nutrition reveals the IUGR swine intestinal development mechanism and Gln the damage mechanism of improving the development of intestine in piglets. In Experiment 1, the miR-29 family was highly expressed in IUGR jejunum (P0.05) by miRNA chip. The target genes of miR-29a predicted by bioinformatics were mainly focused on extracellular matrix and adhesion and connection signal pathway. Western blot analysis showed that IUGR cells of piglets in the extracellular matrix (Fibronectin, Collagen and Integrin beta 1) and tight junction protein (Claudin-1, Occludin and ZO-1) expression was significantly lower than the normal piglets (P0.05), to verify the IUGR damage intestinal extracellular matrix - integrin signal pathway. The Porcine intestinal epithelial cell LINE-1 (IPEC-1) was used as an in vitro model to confirm the inhibitory effect of miR-29a on extracellular matrix, cell adhesion and tight junction gene and protein, thus inhibiting the proliferation and integration of intestinal cells. This study suggests that miR-29a can be used as an important target for detection and target of intestinal mucosal cell development and damage. Test two was designed to investigate the changes in the Gln content in the intestinal tract and the effect of autophagy on the intestinal cell function of IUGR fetal pigs. Fetal porcine jejunum mucosa samples of pregnant D 110, HPLC method for the determination of the free amino acid spectrum and Western blot analysis and IUGR normal fetal pig intestinal autophagy marker microtubule associated protein light chain 3B (Microtubule-associated protein 1 light chain 3B, LC3B) expression changes. The results showed that compared with the normal fetal pigs, the Gln content in the intestinal tract of IUGR fetuses decreased by 66% (P0.01), while the expression level of autophagy marker LC3B- II I was 2 times higher than that of the normal piglets (P0.05). Study on IPEC-1 cell model in vitro showed that Gln deficiency can significantly inhibit the proliferation of IPEC-1 cells were examined by transmission electron microscopy in 0 ~ 8 h with the prolongation of time number of autophagic vacuoles increased gradually (P0.05), the expression of autophagy marker molecules of LC3B- also showed the same trend (P0.05). At the same time, Gln lack of changes in the expression level of mTOR and MAPK signaling pathway related proteins. Gln supplementation can relieve the above phenomenon. Experiment three was designed to investigate the effects of maternal Gln addition on the growth and intestinal development of newborn piglets. By adding 1% Gln to the sow diet at the last 30 days of pregnancy, the average weight (P0.05) of the piglet nests was increased and the coefficient of variation in the nests was reduced (P0.05). By using high performance liquid chromatography and biochemical analysis of sow and newborn piglet plasma free amino acid spectrum and blood biochemical index, to explore the effect of maternal nutritional status and its variation in response to maternal fetal transfer of nutrients added Gln. Analysis of piglets slaughtered showed that the addition of Gln increased the relative intestinal weight (P0.05) of piglets, increased the villi height and width, and the villus height and crypt depth ratio, mucosal thickness and villus surface area (P0.05). Based on the test of four test three, and found that the expression levels of maternal Gln added significantly inhibited miR-29a piglets jejunum tissues (P0.05), promote the extracellular matrix and the expression of tight junction protein (P0.05); at the same time to improve the mTOR of mTOR and its downstream signaling pathway protein phosphorylation of p70S6K (P0.05), suggesting that Gln can promote intestinal mTOR the signal pathway is tightly connected with the enhancement of the cells, and then to maintain intestinal health and promote intestinal function. Combined with 1PEC-1 cell model, the effects of adding different concentrations of Gln on the proliferation of intestinal cells and the expression of related genes and proteins in piglets were investigated. Results: the addition of Gln can promote the proliferation of intestinal epithelial cells and enhance cell monolayer integration (P0.05); the expression and inhibition of miRNA-29a (P0.05), improve the cell extracellular matrix and tight junction protein related gene and protein expression level (P0.05), changed the related transcription factor mRNA expression level. The results suggested that Gln may protect the extracellular matrix through inhibition of miR-29a pathway, and then to maintain intestinal health, promote intestinal development effect. In summary, the results of this study show that miR-29a in IUGR piglets in the high expression of targeted inhibition of extracellular matrix - integrin signaling pathway, Gln deficiency induced autophagy and the change of mTOR and the expression of MAPK signaling pathway; the addition of Gln in late pregnancy in sow diet promoted the expression of piglet growth and intestinal development and protection related protein. To clarify the molecular mechanism of IUGR function of piglet intestinal injury and Gln in intestinal tract of piglets, to promote new growth provides a theoretical basis for further optimization of maternal nutrition program.
【學(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：S828.5
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本文編號：1345552