谷氨酰胺对饥饿小鼠小肠乳脂肪球上皮生长因子E8表达的影响

发布时间：2018-04-15 02:13

本文选题：谷氨酰胺 + 饥饿　；参考：《上海交通大学》2015年硕士论文

【摘要】：目的:乳脂肪球上皮生长因子E8(MFG-E8)是由巨噬细胞和树突状细胞合成分泌的一种可以与磷脂酰丝氨酸及整合素受体结合的糖蛋白。研究指出MFG-E8在维持肠上皮稳态、促进肠黏膜损伤修复中可发挥重要作用,这使得重组MFG-E8作为一种修复肠道损伤的潜在制剂进入人们的视野并引起广泛的关注。然而目前对于MFG-E8的研究主要集中在肠道炎症性损伤方面,对于其在肠道饥饿损伤方面的研究尚未有相关报道。因此我们首先借助小鼠饥饿损伤模型,检测饥饿应激后小鼠小肠黏膜MFG-E8表达的变化情况,探讨MFG-E8在小肠饥饿应激中发挥的作用。其次,作为人体含量最多的一种氨基酸,谷氨酰胺(Gln)可减轻由饥饿导致的肠道损伤,但其具体机制仍不明了。我们通过小鼠饥饿状态下谷氨酰胺灌胃模型检测谷氨酰胺对饥饿小肠黏膜MFG-E8表达的影响,尝试探索谷氨酰胺是否可以通过影响肠道MFG-E8的表达而发挥肠道保护作用。最后,因小肠MFG-E8由固有层巨噬细胞表达,我们在体外细胞培养实验中,通过用不同浓度谷氨酰胺培养小鼠单核巨噬细胞白血病细胞(RAW264.7),检测其对MFG-E8蛋白表达的影响,以对体内试验的结果进行初步的机制探讨。方法:第一部分:首先验证饥饿损伤是否可以改变肠道MFG-E8的表达。通过预实验及相关实验报道,我们得知小鼠在禁食24h后,出现明显的应激行为;48h后活动明显减少;72h后活动已很微弱;84h后小鼠开始死亡。小鼠体重下降在禁食72h后趋于平缓,因此我们选取小鼠体重下降近于最低值而又未死亡的禁食72h的小鼠作为研究对象。将20只雄性C57BL/6小鼠随机分为两组。正常对照组(NC组,10只)正常饮食,自由摄水;全饥饿组(S组,10只)无饲料供给,自由摄水。饥饿72h后脱颈法处死小鼠,取小肠组织。HE染色看小肠绒毛发育情况;TUNEL法检测细胞凋亡;实时荧光定量PCR和蛋白质免疫印迹法检测小肠组织MFG-E8m RNA及MFG-E8蛋白表达。第二部分:测谷氨酰胺对饥饿小鼠小肠MFG-E8表达的影响。将25只雄性C57BL/6小鼠随机分为正常对照组(NC组,n=5),全饥饿组(S组,n=5)和谷氨酰胺灌胃组(G组,共15只)。其中谷氨酰胺灌胃组根据不同的灌胃量(1g/kg/d,3g/kg/d,5g/kg/d)分为3个亚组G1组(n=5)、G3组(n=5)、G5组(n=5)。NC组正常饮食,自由饮水。S组和G组仅自由饮水。G组给予不同剂量谷氨酰胺水溶液灌胃,每天一次,每次0.5ml,共三次。NC组和S组每天给予生理盐水灌胃0.5ml。72h后脱颈法处死小鼠取小肠组织。同第一部分测小肠绒毛发育情况,细胞凋亡,小肠组织MFG-E8m RNA及MFG-E8蛋白表达。第三部分:体外测不同浓度谷氨酰胺对RAW264.7细胞的增殖及对MFG-E8表达的影响。正常啮齿类动物和人类血清谷氨酰胺的浓度约为0.6m M,正常细胞培养及长期储存的谷氨酰胺浓度为2m M,同时研究指出大于等于8m M的谷氨酰胺浓度可诱导多种细胞和组织大量表达热休克蛋白(HSPs),抑制细胞炎症反应。因此我们选用0、0.6、2、10m M的谷氨酰胺孵育RAW264.7细胞,测谷氨酰胺对其增殖以及表达MFG-E8的影响。结果:第一部分:小鼠饥饿72h后小肠绒毛呈现萎缩性改变:黏膜层薄而稀疏,隐窝深度降低,绒毛面积减少,同时伴随着上皮细胞凋亡的增加。MFG-E8在基因水平和蛋白水平表达均增加。第二部分:谷氨酰胺灌胃后小肠绒毛萎缩状态改善,黏膜上皮细胞凋亡减少,尤以3g/kg/d组最为显著。MFG-E8m RNA和蛋白均较单纯饥饿组降低,且m RNA降低与谷氨酰胺灌胃量呈剂量依赖。第三部分:在体外,谷氨酰胺对RAW264.7细胞生长增殖具有重要促进作用。随着谷氨酰胺浓度增高细胞增殖越快。当缺乏谷氨酰胺时,细胞呈现不增长甚至负增长。而其表达MFG-E8蛋白的能力却随着浓度增高而降低,即缺乏谷氨酰胺时,MFG-E8蛋白表达量最高。结论:1、小鼠饥饿应激导致小肠出现损伤性改变:肠绒毛萎缩,肠上皮细胞凋亡增加。而此时出现MFG-E8蛋白及m RNA水平的升高,提示MFG-E8的升高可能是小肠饥饿损伤后的一种自我保护性反应。2、谷氨酰胺灌胃改善了小鼠饥饿应激后出现的小肠绒毛萎缩、上皮细胞凋亡增加,证实谷氨酰胺对于饥饿应激导致的小肠损伤确实有保护作用。小鼠饥饿应激后出现MFG-E8蛋白及m RNA水平增高,而谷氨酰胺灌胃后MFG-E8蛋白及m RNA均较单纯饥饿状态下降低。进一步证明作为一种与肠黏膜损伤修复密切相关的蛋白质,MFG-E8可作为饥饿后反映小肠代偿功能的指标。关注MFG-E8表达的变化,可及时了解肠道的功能状况以采取相应的干预措施。3、体外,细胞在缺乏谷氨酰胺的状态下,巨噬细胞的增殖变弱,而MFG-E8呈现高表达;随着谷氨酰胺浓度的增加,细胞增殖逐渐变强,MFG-E8的表达却降低。提示谷氨酰胺通过影响巨噬细胞的功能而非增加巨噬细胞的数量影响MFG-E8的表达。
[Abstract]:Objective: milk fat globule epidermal growth factor E8 (MFG-E8) is a kind of can be combined with phosphatidylserine and integrin receptor glycoprotein synthesis and secretion of macrophages and dendritic cells. The study indicated that MFG-E8 in the maintenance of intestinal epithelial homeostasis may play an important role in promoting the repair of intestinal mucosa injury, the recombinant MFG-E8 as a repair intestinal injury potential preparations to enter people's vision and attention. However, the research of MFG-E8 mainly concentrated in the intestinal inflammatory injury, for the hungry intestinal injury research has not been reported. So firstly we hunger injury model of mice, the expression of MFG-E8 in intestinal mucosa of mice after starvation stress detection the discussion of MFG-E8 play the role of stress in the small intestine of hunger. Secondly, as the most content of amino acid, glutamine ( Gln) can reduce the intestinal damage caused by hunger, but the specific mechanism is still unclear. We affected by starvation in mice under glutamine gavage model test of glutamine on the expression of MFG-E8 in intestinal mucosa of hunger, try to explore whether glutamine can play gut protective effect by affecting the expression of intestinal MFG-E8. Finally, because of the small intestine MFG-E8 by inherent the expression of macrophage layer, our in vitro experiments, through the cultivation of mouse macrophage leukemia cells with different concentrations of glutamine (RAW264.7), to detect the effect of MFG-E8 protein expression in vivo, on the test results of a preliminary mechanism. Methods: the first part: first verify whether you can change the intestinal injury the expression of hunger MFG-E8. Through the experiment and related reports, we learned that in mice after fasting 24h, stress behavior was 48; H activity decreased significantly; 72h has been very weak; after 84h mice began to die. The decrease of body weight of mice in fasting 72h tends to be flat, so we selected the decrease of body weight of mice to the minimum value of 72h and fasting not died in mice as the research object. 20 male C57BL/6 mice were randomly divided into two groups. The control group (group NC, 10 rats) with normal diet, free water intake; starvation group (group S, 10 rats) without feed supply, free water intake. Hunger after 72h mice were killed by cervical dislocation, small intestine tissue.HE staining at the growth of small intestinal villi; detected cell apoptosis by TUNEL; expression of MFG-E8m RNA and MFG-E8 protein in intestinal tissue by real-time fluorescence quantitative PCR and Western blot. The second part: measuring the effects of glutamine on the expression of MFG-E8 in the small intestine. Hungry mice 25 C57BL/6 male mice were randomly divided into normal control group (group NC, n=5), the hunger group (group S, n=5) And glutamine gavage group (group G, 15 rats). The glutamine gavage group according to the different amount of irrigation stomach (1g/kg/d, 3g/kg/d, 5g/kg/d) were divided into 3 subgroups of G1 group (n=5), G3 group (n=5), group G5 (n=5).NC group, normal diet, free drinking group.S G group and.G group were given only free drinking water solution in different doses orally, once a day, 0.5ml each time, a total of three.NC group and S group were given saline 0.5ml.72h after the mice were killed by cervical small intestine. The same as the first part of measuring the growth of small intestinal villi, cell apoptosis, expression of MFG-E8m RNA and MFG-E8 protein in small intestine. The third part: in vitro test of different concentrations of glutamine on proliferation of RAW264.7 cells and the expression of MFG-E8. The normal rodent animal and human serum glutamine concentration is about 0.6m M, glutamine concentration normal cell culture and long-term storage for 2m M, at the same time to study A large number of heat shock protein expression is greater than the concentration of glutamine is equal to 8m M can be induced by a variety of cells and tissues (HSPs), inhibition of inflammatory reaction. So we use 0,0.6,2,10m for glutamine with M RAW264.7 cells, measuring glutamine on the proliferation and expression of MFG-E8 were investigated. Results: the first part: mice after 72h starvation showed intestinal villi atrophic change: the mucous layer is thin and sparse, crypt depth decreased, villus area decreased, accompanied by increased epithelial apoptosis and expression of.MFG-E8 in gene and protein level were increased. The second part: after intragastric administration of glutamine intestinal villi atrophy improved, apoptosis of epithelial cells decreased, especially in 3g/kg/d group.MFG-E8m RNA and protein were compared with starvation group decreased, and M decreased RNA and glutamine gavage dose dependence. The third part: in vitro, glutamine on RAW264 Has an important role in promoting the growth and proliferation of.7 cells. With the concentration of glutamine increased cell proliferation more quickly. In the absence of glutamine, cells showed no growth or even negative growth. The expression of MFG-E8 protein was decreased with the increasing of concentration, lack of glutamine, MFG-E8 protein expression was the highest. Conclusion: 1. Mice led to starvation stress the small intestine injury changes: intestinal villous atrophy, intestinal epithelial cell apoptosis. The increase of MFG-E8 protein and m RNA levels, suggesting that MFG-E8 may be increased intestinal hunger after injury is a self protective reaction of.2, glutamine improves gastric irrigation in mice after starvation stress villous atrophy, epithelial cells apoptosis, confirmed that indeed glutamine has a protective effect on intestinal injury caused by stress. Hunger after hunger stress in mice MFG-E8 protein and m RNA level increased High glutamine after intragastric administration of MFG-E8 protein and m RNA were compared with starvation decreased. As a further proof of the repair and the intestinal mucosal injury is closely related to the protein, MFG-E8 can be used as the starvation of small intestinal compensatory function index. The changes of the expression of MFG-E8, it is important to understand the function of intestinal tract to take.3. The corresponding intervention measures in vitro, cells in the absence of glutamine state, macrophage proliferation became weak, while MFG-E8 shows high expression; with the increase of glutamine concentration, cell proliferation has been strong, the expression of MFG-E8 was decreased by affecting the function of macrophages that glutamine rather than increase the number of effects of MFG-E8 expression on macrophages.

【学位授予单位】：上海交通大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R459.3

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