谷氨酰胺通过抑制microRNA-29a和细胞自噬调控宫内生长受限仔猪肠道发育的研究

发布时间：2017-12-28 09:41

  本文关键词：谷氨酰胺通过抑制microRNA-29a和细胞自噬调控宫内生长受限仔猪肠道发育的研究　出处：《中国农业大学》2017年博士论文　论文类型：学位论文

  更多相关文章： 宫内生长受限 肠道发育 谷氨酰胺 MicroRNA-29a 自噬 营养 猪

【摘要】：肠道是营养物质消化吸收的主要场所。宫内生长受限(Intrauterine growth restriction,IUGR)可导致仔猪小肠绒毛萎缩,肠道指数下降,细胞骨架蛋白所占比例增加,提示IUGR仔猪肠道细胞增殖与发育受阻。谷氨酰胺(L-Glutamine,Gln)是肠细胞的主要能量来源,在维持正常的肠道屏障功能、调节细胞蛋白质周转以及促进受损肠道的修复中发挥重要作用。且相对于正常胎猪,IUGR胎猪脐带血和羊水中Gln含量较低,推测IUGR胎猪的Gln供给不足是IUGR新生仔猪肠道发育受损的重要原因之一。本研究通过四个试验探讨IUGR新生仔猪肠道中微小RNA(MicroRNA,miRNA)对细胞增殖的调控以及Gln影响细胞自噬的发生,并研究母体添加Gln对新生仔猪生长和肠道发育的影响,分别从分子生物学角度和营养学角度揭示了 IUGR猪肠道发育损伤的机制和Gln改善仔猪肠道发育的机理。试验一通过miRNA芯片筛选出miR-29家族在IUGR仔猪空肠中显著高表达(P0.05),初步生物信息学预测miR-29a的靶基因主要集中在细胞外基质与黏附连接信号通路。Western blot分析发现IUGR仔猪肠道中细胞外基质(Fibronectin、Collagen和Integrin β1)以及紧密连接蛋白(Claudin-1、Occludin和ZO-1)表达量显著低于正常仔猪(P0.05),验证了 IUGR损害肠道胞外基质-整合素信号通路。以新生仔猪空肠上皮细胞系(Porcine intestinal epithelial cell line-1,IPEC-1)为体外模型,证实了 miR-29a对细胞外基质、细胞黏附和紧密连接基因与蛋白的靶向抑制效果,从而抑制肠道细胞增殖与整合性。本研究提示miR-29a可作为肠道黏膜细胞发育与损伤程度的一项重要检测靶标和干预靶标。试验二旨在探讨肠道中Gln含量变化及自噬水平对IUGR胎猪肠道细胞功能的影响。取妊娠d 110的胎猪空肠粘膜样品,高效液相色谱法检测其游离氨基酸谱并Western blot分析正常和IUGR胎猪肠道自噬标志分子微管相关蛋白轻链 3B(Microtubule-associated protein 1 light chain 3B,LC3B)的表达变化。结果表明:与正常胎猪相比,IUGR胎猪肠道中Gln含量下降了 66%(P0.01),而自噬标志分子LC3B-ⅡI的表达水平则为正常仔猪肠道中的2倍(P0.05)。体外IPEC-1细胞模型研究发现Gln缺乏可显著抑制IPEC-1细胞的增殖,透射电镜观察发现在0～8 h内随着处理时间的延长细胞内自噬泡数量逐步增加(P0.05),自噬标志分子LC3B-Ⅱ的表达量也呈现相同趋势(P0.05)。同时,Gln缺乏改变了 mTOR及MAPK信号通路相关蛋白的表达水平。Gln重补充能缓解上述现象发生。试验三旨在探讨母体Gln添加对新生仔猪生长和肠道发育的影响。通过在妊娠期最后30天的母猪饲粮中添加1%的Gln可增加仔猪窝平均个体重(P0.05),降低窝内变异系数(P0.05)。利用高效液相色谱和生化分析技术分析了母猪和新生仔猪血浆游离氨基酸谱和血液生化指标,探讨母体对Gln添加的自身营养状况响应及母胎营养物质转运变化规律。仔猪屠宰分析发现妊娠后期母体添加Gln提高了仔猪相对肠道重量(P0.05),增加了仔猪空肠绒毛高度和宽度、绒毛高度与隐窝深度比、黏膜厚度以及绒毛表面积(P0.05)。试验四在试验三的基础上,发现母体Gln添加显著抑制仔猪空肠组织中miR-29a的表达水平(P0.05),促进胞外基质和紧密连接蛋白表达(P0.05);同时提高mTOR信号通路mTOR及其下游蛋白p70S6K的磷酸化水平(P0.05),提示Gln可促进肠道mTOR信号通路与增强细胞间紧密连接,进而达到维护肠道健康和促进肠道发育的作用。结合1PEC-1细胞模型,探讨添加不同浓度的Gln对仔猪肠道细胞增殖及相关基因和蛋白表达情况的影响。结果发现:添加Gln可促进肠道上皮细胞增殖并且提高单层细胞整合性(P0.05);同时抑制miRNA-29a的表达(P0.05),提高了细胞中胞外基质和紧密连接蛋白等相关的基因和蛋白表达水平(P0.05),改变了相关转录因子的mRNA表达水平,提示Gln有可能通过抑制miR-29a表达保护细胞外基质信号通路,进而达到维护肠道健康、促进肠道发育的作用。综上所述,本研究结果表明在IUGR仔猪肠道中miR-29a的高表达靶向抑制细胞外基质-整合素信号通路,Gln缺乏诱导细胞自噬发生并改变mTOR及MAPK信号通路表达;在妊娠后期母猪饲粮中添加Gln促进了仔猪生长和肠道发育以及相关蛋白表达,阐明了 IUGR仔猪肠道发育损伤的分子机制以及Gln在猪肠道中的保护作用,为进一步优化母体营养方案促进仔猪生长提供了新的理论依据。
[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.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S828.5
，

本文编号：1345552