本文選題：腸道共生菌 + 枯否細(xì)胞��； 參考：《中國科學(xué)技術(shù)大學(xué)》2015年博士論文

【摘要】：哺乳動物的腸道內(nèi)存在著種類和數(shù)量繁多的共生菌群,它們既可以被免疫系統(tǒng)所調(diào)控,又可以影響免疫系統(tǒng)細(xì)胞的組成比例和免疫應(yīng)答反應(yīng)。腸道共生菌群不僅參與調(diào)控腸道內(nèi)的免疫系統(tǒng)應(yīng)答以及腸道相關(guān)的疾病,也可以參與調(diào)控肺臟、肝臟和皮膚等身體其它器官的免疫應(yīng)答反應(yīng)以及相關(guān)的炎癥。來源于腸道共生菌群的代謝產(chǎn)物以及食物抗原可以通過門靜脈直接進(jìn)入肝臟,對肝臟的炎癥和免疫應(yīng)答進(jìn)行調(diào)控。腸道共生菌群被發(fā)現(xiàn)可以參與肝癌、自身免疫性肝炎以及非酒精性脂肪肝的發(fā)生過程,也被認(rèn)為可以促進(jìn)肝臟再生的過程,然而其中的免疫學(xué)機(jī)制目前研究得并不是很清楚。 在本文的研究中,我們在小鼠的飲用水中分別加入氨芐霉素、萬古霉素、硫酸新霉素以及甲硝唑四種抗生素,給小鼠喂水一個月后清除腸道的共生菌群,將飲用正常水的小鼠作為其相應(yīng)的對照,進(jìn)行肝切除手術(shù),通過觀察小鼠的肝體比重、免疫組化的方法檢測小鼠肝細(xì)胞增殖的速率來比較兩種小鼠肝臟再生的能力。進(jìn)一步給喂水一周、二周、三周、四周的小鼠分別進(jìn)行肝切除以及檢測結(jié)腸中細(xì)菌載量,來分析比較腸道共生菌群的載量與肝臟再生能力之間的關(guān)系,再分別將氨芐霉素、萬古霉素、硫酸新霉素以及甲硝唑四種抗生素分別加入小鼠的飲用水中,探究何種抗生素敏感的共生菌群影響了肝臟再生的能力。通過比較抗菌素處理小鼠和對照小鼠在肝切除的不同時間點免疫系統(tǒng)細(xì)胞的比例和絕對數(shù)量來分析腸道共生菌群是通過影響免疫系統(tǒng)的亞群來調(diào)控肝臟再生的過程。 我們的研究結(jié)果發(fā)現(xiàn)口服抗生素清除腸道共生菌群后小鼠肝臟ALT、AST水平?jīng)]有發(fā)生顯著上調(diào),也不會引起小腸和結(jié)腸的炎癥發(fā)生,小鼠各個時間的肝體比重以及肝細(xì)胞的增長速率較對照組明顯下調(diào),隨著抗生素喂水時間的延長,小鼠結(jié)腸中的細(xì)菌載量也相應(yīng)的減少,伴隨著小鼠肝臟再生能力的下降,在三周后穩(wěn)定下來。進(jìn)一步,我們分別將這四種抗生素加入到小鼠的飲用水中,觀察單獨一種抗生素對于小鼠肝臟再生能力的影響,結(jié)果發(fā)現(xiàn)氨芐霉素的處理相比于其它三種抗生素,對肝臟再生的抑制能力最強(qiáng),暗示著氨芐霉素敏感的腸道共生菌群可以促進(jìn)肝臟再生的過程。 對免疫學(xué)機(jī)制的研究發(fā)現(xiàn),在抗生素喂水小鼠中肝切除的各個時間點,肝臟CD3+NK1.1+的NKT細(xì)胞比例、數(shù)量以及活化程度較正常飲用水小鼠相比顯著增加,并且這群增加的NKT細(xì)胞是CDld依賴的,高表達(dá)趨化因子CXCR6且凋亡比例顯著減少,在抗原刺激后活化性受體NKG2D, Fas, CD69的表達(dá)顯著上調(diào),抑制性受體NKG2A的表達(dá)上調(diào),分泌細(xì)胞因子IFNγ的能力顯著增強(qiáng)�？股匚顾∈笤贑D1d依賴的NKT細(xì)胞缺失或者阻斷CD1d分子的功能的時候肝細(xì)胞的增殖能力增強(qiáng),而轉(zhuǎn)輸CD1d依賴的NKT細(xì)胞會加重肝細(xì)胞增殖能力的減弱。T細(xì)胞以及NK細(xì)胞的清除小鼠中在抗生素口服處理后肝臟再生的能力無明顯變化。 接下來我們研究發(fā)現(xiàn),抗生素處理的小鼠中Kupffer細(xì)胞在肝臟中聚集、活化,并且Kupffer細(xì)胞在肝臟中的聚集是由于自身增殖能力增強(qiáng)而并非由外周血單個核細(xì)胞分化而來。Kupffer細(xì)胞表面活化性的標(biāo)志CD80, CD86以及CD68的表達(dá)顯著上調(diào),TLR信號受體TLR2, TLR4的表達(dá)也明顯上調(diào)。在體外LPS的刺激作用下,其分泌IL-6,TNFα, IL-10以及IL-12的能力顯著增強(qiáng),TLR3的激動劑polyI:C可以明顯回復(fù)抗生素處理小鼠肝臟再生的能力,而單次腹腔注射TLR2的激動劑Pamsck4以及TLR4的激動劑LPS顯著減弱抗生素處理小鼠肝臟再生能力。接下來我們連續(xù)三天給小鼠腹腔注射低劑量的LPS模擬小鼠LPS耐受誘導(dǎo)模型,研究發(fā)現(xiàn),LPS耐受模型可以顯著下調(diào)抗生素處理小鼠Kupffer細(xì)胞表面CD80,CD86以及TLR受體的表達(dá),而相應(yīng)的肝臟再生的能力顯著上調(diào)。 最后,我們在用脂質(zhì)體體內(nèi)清除Kupffer細(xì)胞,發(fā)現(xiàn)清除Kupffer:細(xì)胞,肝切除12小時后,肝臟NKT細(xì)胞的活化性受體NKG2D, CD69和CD25的表達(dá)顯著下調(diào),分泌細(xì)胞因子IFNγ的能力減弱,同時我們在抗生素喂水小鼠肝切除前后的血清中檢測到了IL-12表達(dá)的顯著上調(diào),IL-12p40缺陷的小鼠或者IL-12的中和抗體可以顯著抑制肝臟中的NKT細(xì)胞的活化,進(jìn)一步促進(jìn)抗生素喂水小鼠肝實質(zhì)細(xì)胞的增殖。 綜上所述,我們可以推斷腸道共生菌群是通過維持肝臟Kupffer細(xì)胞處于耐受穩(wěn)態(tài),防止NKT細(xì)胞的過度活化來促進(jìn)肝臟再生的過程,我們的數(shù)據(jù)暗示著長時間抗生素的使用,會破壞腸道共生菌群,最后通過影響肝細(xì)胞再生過程來影響肝臟的功能。
[Abstract]:The intestinal commensal bacteria group is not only involved in regulating immune system response and intestinal related diseases in the intestinal tract , but also can regulate the immune response reaction of other organs such as lung , liver and skin , and regulate the inflammation and immune response of the liver . The intestinal symbiotic bacteria group is found to be involved in the process of liver cancer , autoimmune hepatitis and non - alcoholic fatty liver , and the immunological mechanism is not very clear at present .

In this study , we used four kinds of antibiotics of ampicillin , vancomycin , new sulfate and metronidazole respectively in the drinking water of mice . The mice were fed with water for one month to remove the symbiotic bacteria in the intestinal tract .

The results showed that there was no significant increase in ALT and AST levels in the liver of mice after oral antibiotic removal , but also did not result in the inflammation of small intestine and colon .

The expression of NKG2D , Fas and CD69 increased significantly , and the expression of activated receptor NKG2D , Fas and CD69 increased significantly after anti - primary stimulation .

The expression of IL - 6 , TNF.alpha . , IL - 10 and IL - 12 was significantly increased in mice treated with antibiotics . The expression of IL - 6 , TNF - 偽 , IL - 10 and IL - 12 increased significantly in the liver .

Finally , we found that the expression of NKG2D , CD69 and CD25 in liver NKT cells was down - regulated after 12 hours after liver resection , and the ability to secrete cytokines IFN緯 was decreased . At the same time , the expression of IL - 12 was detected in the serum before and after liver resection of antibiotic - fed mice , and the neutralizing antibody of IL - 12p40 - deficient mice or IL - 12 could significantly inhibit the activation of NKT cells in the liver and further promote the proliferation of the liver parenchyma cells of antibiotic - fed mice .

In conclusion , we can conclude that the intestinal commensal flora is the process of promoting the regeneration of liver by maintaining the stable state and preventing the excessive activation of NKT cells . Our data suggest that the use of long - term antibiotics can destroy the flora of intestinal bacteria , and finally influence the liver function by influencing the regeneration process of liver cells .
【學(xué)位授予單位】：中國科學(xué)技術(shù)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：Q418

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 ;Isolation of Lymphocytes and Their Innate Immune Characterizations from Liver,Intestine,Lung and Uterus[J];Cellular & Molecular Immunology;2005年04期

2 ;The Roles of Innate Immune Cells in Liver Injury and Regeneration[J];Cellular & Molecular Immunology;2007年04期

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本文編號：2005813