本文選題：谷氨酰胺 + 饑餓　； 參考：《上海交通大學(xué)》2015年碩士論文

【摘要】：目的:乳脂肪球上皮生長因子E8(MFG-E8)是由巨噬細(xì)胞和樹突狀細(xì)胞合成分泌的一種可以與磷脂酰絲氨酸及整合素受體結(jié)合的糖蛋白。研究指出MFG-E8在維持腸上皮穩(wěn)態(tài)、促進(jìn)腸黏膜損傷修復(fù)中可發(fā)揮重要作用,這使得重組MFG-E8作為一種修復(fù)腸道損傷的潛在制劑進(jìn)入人們的視野并引起廣泛的關(guān)注。然而目前對于MFG-E8的研究主要集中在腸道炎癥性損傷方面,對于其在腸道饑餓損傷方面的研究尚未有相關(guān)報(bào)道。因此我們首先借助小鼠饑餓損傷模型,檢測饑餓應(yīng)激后小鼠小腸黏膜MFG-E8表達(dá)的變化情況,探討MFG-E8在小腸饑餓應(yīng)激中發(fā)揮的作用。其次,作為人體含量最多的一種氨基酸,谷氨酰胺(Gln)可減輕由饑餓導(dǎo)致的腸道損傷,但其具體機(jī)制仍不明了。我們通過小鼠饑餓狀態(tài)下谷氨酰胺灌胃模型檢測谷氨酰胺對饑餓小腸黏膜MFG-E8表達(dá)的影響,嘗試探索谷氨酰胺是否可以通過影響腸道MFG-E8的表達(dá)而發(fā)揮腸道保護(hù)作用。最后,因小腸MFG-E8由固有層巨噬細(xì)胞表達(dá),我們在體外細(xì)胞培養(yǎng)實(shí)驗(yàn)中,通過用不同濃度谷氨酰胺培養(yǎng)小鼠單核巨噬細(xì)胞白血病細(xì)胞(RAW264.7),檢測其對MFG-E8蛋白表達(dá)的影響,以對體內(nèi)試驗(yàn)的結(jié)果進(jìn)行初步的機(jī)制探討。方法:第一部分:首先驗(yàn)證饑餓損傷是否可以改變腸道MFG-E8的表達(dá)。通過預(yù)實(shí)驗(yàn)及相關(guān)實(shí)驗(yàn)報(bào)道,我們得知小鼠在禁食24h后,出現(xiàn)明顯的應(yīng)激行為;48h后活動明顯減少;72h后活動已很微弱;84h后小鼠開始死亡。小鼠體重下降在禁食72h后趨于平緩,因此我們選取小鼠體重下降近于最低值而又未死亡的禁食72h的小鼠作為研究對象。將20只雄性C57BL/6小鼠隨機(jī)分為兩組。正常對照組(NC組,10只)正常飲食,自由攝水;全饑餓組(S組,10只)無飼料供給,自由攝水。饑餓72h后脫頸法處死小鼠,取小腸組織。HE染色看小腸絨毛發(fā)育情況;TUNEL法檢測細(xì)胞凋亡;實(shí)時熒光定量PCR和蛋白質(zhì)免疫印跡法檢測小腸組織MFG-E8m RNA及MFG-E8蛋白表達(dá)。第二部分:測谷氨酰胺對饑餓小鼠小腸MFG-E8表達(dá)的影響。將25只雄性C57BL/6小鼠隨機(jī)分為正常對照組(NC組,n=5),全饑餓組(S組,n=5)和谷氨酰胺灌胃組(G組,共15只)。其中谷氨酰胺灌胃組根據(jù)不同的灌胃量(1g/kg/d,3g/kg/d,5g/kg/d)分為3個亞組G1組(n=5)、G3組(n=5)、G5組(n=5)。NC組正常飲食,自由飲水。S組和G組僅自由飲水。G組給予不同劑量谷氨酰胺水溶液灌胃,每天一次,每次0.5ml,共三次。NC組和S組每天給予生理鹽水灌胃0.5ml。72h后脫頸法處死小鼠取小腸組織。同第一部分測小腸絨毛發(fā)育情況,細(xì)胞凋亡,小腸組織MFG-E8m RNA及MFG-E8蛋白表達(dá)。第三部分:體外測不同濃度谷氨酰胺對RAW264.7細(xì)胞的增殖及對MFG-E8表達(dá)的影響。正常嚙齒類動物和人類血清谷氨酰胺的濃度約為0.6m M,正常細(xì)胞培養(yǎng)及長期儲存的谷氨酰胺濃度為2m M,同時研究指出大于等于8m M的谷氨酰胺濃度可誘導(dǎo)多種細(xì)胞和組織大量表達(dá)熱休克蛋白(HSPs),抑制細(xì)胞炎癥反應(yīng)。因此我們選用0、0.6、2、10m M的谷氨酰胺孵育RAW264.7細(xì)胞,測谷氨酰胺對其增殖以及表達(dá)MFG-E8的影響。結(jié)果:第一部分:小鼠饑餓72h后小腸絨毛呈現(xiàn)萎縮性改變:黏膜層薄而稀疏,隱窩深度降低,絨毛面積減少,同時伴隨著上皮細(xì)胞凋亡的增加。MFG-E8在基因水平和蛋白水平表達(dá)均增加。第二部分:谷氨酰胺灌胃后小腸絨毛萎縮狀態(tài)改善,黏膜上皮細(xì)胞凋亡減少,尤以3g/kg/d組最為顯著。MFG-E8m RNA和蛋白均較單純饑餓組降低,且m RNA降低與谷氨酰胺灌胃量呈劑量依賴。第三部分:在體外,谷氨酰胺對RAW264.7細(xì)胞生長增殖具有重要促進(jìn)作用。隨著谷氨酰胺濃度增高細(xì)胞增殖越快。當(dāng)缺乏谷氨酰胺時,細(xì)胞呈現(xiàn)不增長甚至負(fù)增長。而其表達(dá)MFG-E8蛋白的能力卻隨著濃度增高而降低,即缺乏谷氨酰胺時,MFG-E8蛋白表達(dá)量最高。結(jié)論:1、小鼠饑餓應(yīng)激導(dǎo)致小腸出現(xiàn)損傷性改變:腸絨毛萎縮,腸上皮細(xì)胞凋亡增加。而此時出現(xiàn)MFG-E8蛋白及m RNA水平的升高,提示MFG-E8的升高可能是小腸饑餓損傷后的一種自我保護(hù)性反應(yīng)。2、谷氨酰胺灌胃改善了小鼠饑餓應(yīng)激后出現(xiàn)的小腸絨毛萎縮、上皮細(xì)胞凋亡增加,證實(shí)谷氨酰胺對于饑餓應(yīng)激導(dǎo)致的小腸損傷確實(shí)有保護(hù)作用。小鼠饑餓應(yīng)激后出現(xiàn)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均較單純饑餓狀態(tài)下降低。進(jìn)一步證明作為一種與腸黏膜損傷修復(fù)密切相關(guān)的蛋白質(zhì),MFG-E8可作為饑餓后反映小腸代償功能的指標(biāo)。關(guān)注MFG-E8表達(dá)的變化,可及時了解腸道的功能狀況以采取相應(yīng)的干預(yù)措施。3、體外,細(xì)胞在缺乏谷氨酰胺的狀態(tài)下,巨噬細(xì)胞的增殖變?nèi)?而MFG-E8呈現(xiàn)高表達(dá);隨著谷氨酰胺濃度的增加,細(xì)胞增殖逐漸變強(qiáng),MFG-E8的表達(dá)卻降低。提示谷氨酰胺通過影響巨噬細(xì)胞的功能而非增加巨噬細(xì)胞的數(shù)量影響MFG-E8的表達(dá)。
[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.

【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R459.3

【相似文獻(xiàn)】

相關(guān)期刊論文 前1條

1 周明華;趙麗萍;張麗雯;;培養(yǎng)中E8雞胚前腦5-羥色胺免疫反應(yīng)神經(jīng)細(xì)胞的鑒定[J];解剖學(xué)報(bào);1990年04期

相關(guān)重要報(bào)紙文章 前1條

1 記者 成淇平;馬來西亞E8公司捐贈污水處理技術(shù)[N];云南日報(bào);2008年

相關(guān)碩士學(xué)位論文 前3條

1 張利娟;谷氨酰胺對饑餓小鼠小腸乳脂肪球上皮生長因子E8表達(dá)的影響[D];上海交通大學(xué);2015年

2 石麗娜;桃遺傳轉(zhuǎn)化再生體系的建立及果實(shí)特異性基因E8啟動子的克隆[D];甘肅農(nóng)業(yè)大學(xué);2007年

3 王玉霞;果實(shí)特異性啟動子E8和桃反義PG基因的克隆及序列分析[D];甘肅農(nóng)業(yè)大學(xué);2008年

，

本文編號：1752057