發(fā)布時(shí)間：2018-07-10 05:49

  本文選題：非酒精性脂肪肝 + NLRP3�。� 參考：《重慶醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的構(gòu)建非酒精性脂肪肝小鼠模型,探討心磷脂激活NLRP3炎癥小體而促進(jìn)非酒精性脂肪肝病變的機(jī)制。方法1.分別使用高脂飼料和低脂飼料喂養(yǎng)C57BL/6J小鼠各10只,16周后取肝臟組織,采用油紅O染色、HE染色、免疫組化染色及血清肝功能測(cè)定,評(píng)估非酒精性脂肪肝模型建立狀況。2.分別提取非酒精性脂肪肝模型組和對(duì)照組小鼠肝組織的總蛋白,采用Western Blot技術(shù)比較兩組標(biāo)本NLRP3的表達(dá)水平。3.分離培養(yǎng)C57BL/6J小鼠Kupffer細(xì)胞。分別用棕櫚酸(模擬體內(nèi)游離脂肪酸在Kupffer細(xì)胞沉積)和生理鹽水(對(duì)照)刺激兩組Kupffe細(xì)胞后,采用Western Blot比較兩組的NLRP3表達(dá)水平;ELISA測(cè)定細(xì)胞培養(yǎng)液上清液的IL-1β水平;用帶His標(biāo)簽的NLRP3過表達(dá)質(zhì)粒轉(zhuǎn)染Kupffer細(xì)胞,將細(xì)胞培養(yǎng)48h后提取蛋白,用protein-lipid overlay技術(shù)檢測(cè)心磷脂與NLRP3蛋白結(jié)合情況。結(jié)果1.高脂飲食組較低脂飲食組小鼠的肝臟體積增大、肝細(xì)胞脂肪變性顯著,Kupffer細(xì)胞明顯增生,灶性炎癥細(xì)胞浸潤(rùn),外周血ALT顯著增高(高脂組ALT為134.3U/L,低脂組為26U/L)。2.非酒精性脂肪肝模型組小鼠NLRP3炎癥小體表達(dá)水平較對(duì)照組增高了42.16%。3.棕櫚酸刺激的Kupffer細(xì)胞上清較對(duì)照組Kupffer細(xì)胞的上清含有更多的IL-1β,分別是169.3±6.8 pg/ml and134.6±2.4 pg/ml respectively(p0.01)。棕櫚酸刺激組NLRP3蛋白表達(dá)水平較對(duì)照組高103.68%;c-IL-1β蛋白水平也較對(duì)照組高38.78%。將Kupffer細(xì)胞總蛋白孵育PIP strip膜,再用抗His抗體雜交,含心磷脂的孔出現(xiàn)免疫印跡。結(jié)論1.非酒精性脂肪肝模型構(gòu)建成功。2.NLRP3炎癥小體在非酒精性脂肪肝組織中表達(dá)上調(diào),提示其可能參與非酒精性脂肪肝的病變進(jìn)程。3.棕櫚酸刺激的Kupffer細(xì)胞NLRP3和c-IL-1β表達(dá)水平均明顯增高。心磷脂能與Kupffer細(xì)胞中NLRP3炎癥小體結(jié)合并將其激活,可能促進(jìn)非酒精性脂肪肝的病理進(jìn)程。
[Abstract]:Objective to construct a nonalcoholic fatty liver mouse model and explore the mechanism of cardiolipin activation of NLRP3 inflammatory body to promote nonalcoholic fatty liver disease. Methods 1. 10 C57BL/6J mice were fed with high fat diet and low fat diet respectively. After 16 weeks, liver tissue was taken by oil red O staining, HE staining, immunohistochemical staining and serum liver function test. To evaluate the establishment of non-alcoholic fatty liver model (.2.), the total protein of the liver tissues of the nonalcoholic fatty liver model group and the control group was extracted respectively. The expression level of NLRP3 in the two groups was compared with the C57BL/6J Blot technique by using the Western Blot technique to isolate the C57BL/6J mice Kupffer cells. After two groups of Kupffe cells were stimulated by cell deposition and normal saline (control), the expression level of NLRP3 in two groups was compared with Western Blot; IL-1 beta level in supernatant of cell culture liquid was measured by ELISA; Kupffer cells were transfected with NLRP3 overexpressed plasmid with His label, the cells were cultured for 48H and extracted protein, and the cardiac phosphorus was detected by protein-lipid overlay technique. Results in 1. high fat diet group, the liver volume increased, the liver cell fatty degeneration was significant, the Kupffer cells were obviously proliferated, the focal inflammatory cells were infiltrated, the peripheral blood ALT was significantly increased (high fat group ALT was 134.3U/L, the low fat group was 26U/ L).2. non-alcoholic fatty liver model group mice NLRP3 inflammation small Compared with the control group, the level of 42.16%.3. palmitic acid stimulated Kupffer cell supernatant was more IL-1 beta than that of the control group, which was 169.3 + 6.8 pg/ml and134.6 + 2.4 pg/ml respectively (P0.01). The expression level of NLRP3 protein in the palmitic acid stimulation group was 103.68% higher than that in the control group, and the c-IL-1 beta protein level was also higher than that of the control group. The total protein of Kupffer cell was incubated with the total protein of PIP strip membrane in the group of high 38.78%., and then hybridized with anti His antibody, and the pore of cardiac phospholipid appeared to be immunoblotting. Conclusion 1. non-alcoholic fatty liver model was successfully constructed and the expression of.2.NLRP3 inflammatory body was up-regulated in non-alcoholic fatty liver tissue, suggesting that it could be involved in the process of non-alcoholic fatty liver disease,.3. brown. The expression level of NLRP3 and c-IL-1 beta in Kupffer cells stimulated by acid was significantly increased. Cardiolipin could be combined with NLRP3 inflammatory bodies in Kupffer cells and activated it, which may promote the pathological process of non-alcoholic fatty liver.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R575

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 程伯基;;心磷脂和線粒體內(nèi)膜[J];生理科學(xué)進(jìn)展;1987年01期

2 紀(jì)紹梅,辜清吾;從牛心中分離和純化心磷脂(摘要)[J];中國(guó)皮膚性病學(xué)雜志;1992年04期

3 程昆蓉,程伯基,施明連,林克椿;心磷脂電化學(xué)行為的研究[J];生物化學(xué)雜志;1992年06期

4 楊林西,葛建國(guó),臧宏;柱層析法分離與純化心磷脂[J];蘭州醫(yī)學(xué)院學(xué)報(bào);1999年04期

5 程昆蓉,程伯基,宋慧芳,喬梁,王動(dòng),倪雪梅,林克椿;細(xì)胞色素C引起心磷脂的還原[J];生物化學(xué)雜志;1992年05期

6 程伯基,喬梁,宋林野,張鳳立,董玉枝,林克椿;阿霉素對(duì)細(xì)胞色素C誘導(dǎo)心磷脂多形性相變的影響[J];北京醫(yī)科大學(xué)學(xué)報(bào);1989年05期

7 程伯基,董玉枝,喬玉蘭,林克椿,虞群,葉建平,壽涵森;細(xì)胞色素C和含心磷脂的人工脂膜相互作用[J];生物化學(xué)雜志;1990年04期

8 龔政;熊源長(zhǎng);;細(xì)胞色素C/心磷脂復(fù)合體和氧化心磷脂及細(xì)胞凋亡[J];細(xì)胞與分子免疫學(xué)雜志;2008年10期

9 裴瑾,陳星海,楊翰儀;硅膠G層析法提取心磷脂及其鑒定[J];白求恩醫(yī)科大學(xué)學(xué)報(bào);1997年06期

10 程昆蓉,程伯基,沈子威,林克椿;心磷脂-細(xì)胞色素C體系CD譜的研究[J];生物化學(xué)雜志;1992年03期

相關(guān)會(huì)議論文 前2條

1 趙寶貞;吳玉薇;魯崎唔;黃芬;;膽固醇對(duì)心磷脂—磷酯酰絲氨酸脂質(zhì)體相變行為的影響[A];第五次全國(guó)電子顯微學(xué)會(huì)議論文摘要集[C];1988年

2 沈曉蕾;趙志剛;袁慧娟;馬躍華;張會(huì)峰;田睿;;糖尿病冠心病心磷脂病態(tài)重構(gòu)的分子機(jī)制[A];中華醫(yī)學(xué)會(huì)第十二次全國(guó)內(nèi)分泌學(xué)學(xué)術(shù)會(huì)議論文匯編[C];2013年

相關(guān)博士學(xué)位論文 前1條

1 李佳;心磷脂�；D(zhuǎn)移酶1對(duì)線粒體形態(tài)和功能的分子調(diào)控[D];西北農(nóng)林科技大學(xué);2012年

相關(guān)碩士學(xué)位論文 前4條

1 李亞坤;心磷脂�；D(zhuǎn)移酶1在糖尿病模型大鼠坐骨神經(jīng)中的表達(dá)及影響[D];鄭州大學(xué);2016年

2 汪濤;心磷脂激活NLRP3炎癥小體促進(jìn)非酒精性脂肪肝的機(jī)制研究[D];重慶醫(yī)科大學(xué);2017年

3 沈曉蕾;不同慢性疾病中的心磷脂代謝變化及意義[D];河南大學(xué);2013年

4 陳燕;電解質(zhì)溶液中心磷脂的自組裝形態(tài)及結(jié)構(gòu)穩(wěn)定性[D];復(fù)旦大學(xué);2009年

，

本文編號(hào)：2112307