本文關(guān)鍵詞：膿毒癥大鼠小腸上皮自噬水平及腦腸肽Ghrelin對(duì)其調(diào)節(jié)作用的研究　出處：《蘭州大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 盲腸結(jié)扎穿刺術(shù) 膿毒癥 小腸上皮 自噬 盲腸結(jié)扎穿刺術(shù) 膿毒癥 Ghrelin 小腸上皮 自噬

【摘要】：第一部分：膿毒癥大鼠小腸上皮細(xì)胞的自噬水平研究 目的：研究盲腸結(jié)扎穿刺(Cecal ligation and puncture,CLP)誘導(dǎo)的膿毒癥大鼠小腸上皮自噬水平的變化,探討自噬與膿毒癥腸上皮損害的關(guān)系。 方法：雄性SD大鼠60只隨機(jī)分成對(duì)照組,CLP4h組、CLP8h組、CLP12h組、CLP16h組及CLP20h組(n=10)。建立大鼠盲腸結(jié)扎穿刺模型后,在各時(shí)間點(diǎn)隨機(jī)處死大鼠,收集大鼠血清及小腸腸段,低溫下刮取小腸上皮粘膜保存待檢。酶聯(lián)免疫吸附法檢測(cè)血清及粘膜細(xì)胞因子變化,Western-blot法及Real time-PCR法從蛋白及基因水平檢測(cè)粘膜組織中自噬相關(guān)蛋白LC3表達(dá)量,統(tǒng)計(jì)學(xué)分析各組間差異；透射電子顯微鏡觀察膿毒癥8h和20h大鼠腸上皮細(xì)胞微細(xì)胞結(jié)構(gòu)變化及自噬泡形態(tài),并統(tǒng)計(jì)自噬泡及自噬溶酶體數(shù)量。 結(jié)果：CLP誘導(dǎo)的膿毒癥大鼠血清及粘膜細(xì)胞因子TNF-a和MCP-1在4h即升高,明顯高于對(duì)照組(P0.05),自噬相關(guān)蛋白LC3-Ⅱ/LC3在膿毒癥8h明顯高于對(duì)照組(1.255±0.088vs1.470±0.295,P=0.038,n=4),12h后下降；LC3基因轉(zhuǎn)錄水平較對(duì)照組升高(0.120±0.069vs3.104±1.191,P=0.00,n=10),隨之下降。電鏡觀察CLP8h和20h腸上皮細(xì)胞,線粒體損傷明顯,腫脹溶解,內(nèi)質(zhì)網(wǎng)空泡化,CLP8h小腸上皮細(xì)胞內(nèi)自噬泡和自噬溶酶體數(shù)量較20h組明顯增多(19.33±2.16vs9.33+1.86,37.67±3.27vs19.50±1.87,P=0.00,n=3),CLP20h細(xì)胞內(nèi)也可見較大的自噬溶酶體。 結(jié)論：膿毒癥大鼠小腸局部及全身炎性因子TNF-a早期達(dá)高峰,小腸粘膜自噬相關(guān)蛋白LC3在蛋白和基因水平表達(dá)早期升高,隨后下降。自噬在膿毒癥病理環(huán)境下受到炎性細(xì)胞因子變化影響,與TNF-a水平正相關(guān)。 第二部分：腦腸肽Ghrelin對(duì)膿毒癥大鼠腸上皮細(xì)胞自噬的調(diào)節(jié)作用 目的：研究腦腸肽Ghrelin對(duì)膿毒癥大鼠腸上皮自噬的調(diào)節(jié)作用,探討Ghrelin通過調(diào)節(jié)細(xì)胞自噬對(duì)膿毒癥大鼠腸道發(fā)揮保護(hù)作用。 方法：將40只雄性SD大鼠隨機(jī)分成正常組、假手術(shù)組、CLP20h組及Ghrelin干預(yù)組,通過大鼠盲腸結(jié)扎穿刺術(shù)建立膿毒癥動(dòng)物模型,并經(jīng)大鼠尾靜脈持續(xù)泵注Ghrelin20h,隨后處死各組動(dòng)物,收集大鼠血清及小腸組織；截取部分小腸段固定后待行病理及電鏡觀察,另一部分小腸在低溫下刮取腸粘膜待行分子生物學(xué)檢測(cè)。光學(xué)顯微鏡觀察各組小腸上皮的病理改變,透射電子顯微鏡觀察小腸上皮微細(xì)胞結(jié)構(gòu)變化及細(xì)胞內(nèi)自噬泡形態(tài)和數(shù)量。免疫熒光觀察小腸上皮細(xì)胞內(nèi)GFP-LC3表達(dá)水平。Western-blot及Real-time PCR檢測(cè)小腸粘膜自噬相關(guān)蛋白LC3水平,酶聯(lián)免疫吸附法檢測(cè)各組動(dòng)物血清和粘膜內(nèi)炎癥因子水平的變化,統(tǒng)計(jì)學(xué)分析各組間差異。 結(jié)果：(1)與正常組及假手術(shù)組相比,膿毒癥20h大鼠腸上皮病理損傷明顯,血清及粘膜TNF-a(154.57±19.54vs88.58±8.19、96.08±9.93,74.95±4.89vs19.94±1.10、22.44±4.18,P=0.00,n=10)和MCP-1(96.33±4.89vs61.66±6.94、69.16±10.42,66.74±4.80vs41.74±1.78、49.24±4.32,P=0.00,n=10)水平升高,LC3蛋白表達(dá)升高(1.52±0.03vs0.98±0.18、1.25±0.09,P=0.00,n=4),其基因轉(zhuǎn)錄水平也升高(0.42±0.16vs0.13±0.11、0.13±0.12,P=0.00,n=10)。膿毒癥大鼠小腸上皮GFP-LC3表達(dá)較假手術(shù)組升高(210±18.03vs168±17.66,P=0.003,n=5)。小腸上皮細(xì)胞自噬泡及自噬溶酶體數(shù)量較假手術(shù)組升高(36.83±3.76vs9.33±1.86,71.83±5.27vs19.50±1.87,P=0.00,n=3)。(2)與膿毒癥組比較,小腸上皮粘膜毛細(xì)血管充血減輕。血清及腸粘膜組織TNF-a(154.57±19.54vs192.75±21.91,74.95±4.89vs96.85±15.35,P=0.00,n=10)和MCP-1(96.33±4.89vs189.93±14.59,66.74±4.80vs99.08±7.47,P=0.00,n=10)降低,LC3蛋白表達(dá)升高(1.52±0.03vs1.35±0.06,P=0.035,n=4),LC3基因轉(zhuǎn)錄水平(0.42±0.16vs0.14±0.10,P=0.00,n=10)。腸上皮細(xì)胞GFP-LC3表達(dá)增多(210±18.03vs177.80±17.66,P=0.014,n=5)。Ghrelin組腸上皮細(xì)胞內(nèi)自噬泡及自噬溶酶體數(shù)量明顯升高(36.83±3.76vs19.33±2.16,71.83±5.27vs37.67±3.27,P=0.00,n=3)。 結(jié)論：腦腸肽Ghrelin明顯減輕膿毒癥大鼠小腸上皮病理損害,降低局部及全身炎癥因子水平,促進(jìn)小腸上皮細(xì)胞自噬相關(guān)蛋白LC3表達(dá)及轉(zhuǎn)錄,增加細(xì)胞內(nèi)自噬泡及自噬溶酶體數(shù)量,具有上調(diào)膿毒癥大鼠小腸上皮細(xì)胞自噬的作用。
[Abstract]:The first part: Study on the autophagy level of small intestinal epithelial cells in sepsis rats
Objective: To investigate the changes of autophagy level in intestinal epithelium of sepsis induced by Cecal ligation and puncture (CLP), and to explore the relationship between autophagy and intestinal epithelial lesion in sepsis.
Methods: 60 male SD rats were randomly divided into control group, CLP4h group, CLP8h group, CLP12h group, CLP16h group and CLP20h group (n=10). To establish a rat model of cecal ligation and puncture after rats were sacrificed randomly at each time point, collect serum and intestinal segment, scraping intestinal epithelium under low temperature save the mucosa to be detected. Enzyme linked immunosorbent assay in serum and mucosal changes of cytokines, the expression of autophagy from protein and gene level detection of mucosa associated protein LC3 in Western-blot method and Real time-PCR method, statistical analysis of differences between groups; transmission electron microscopy observation of intestinal epithelial cells in sepsis rats 8h and 20h micro cell structure and the change of autophagy and autophagic vacuole morphology, statistical and autophasosomes number.
Results: CLP induced sepsis in rat serum and mucosal cytokines TNF-a and MCP-1 increased in 4h, significantly higher than the control group (P0.05), autophagy related protein LC3- II /LC3 in sepsis and 8h was significantly higher than the control group (1.255 + 0.088vs1.470 + 0.295, P=0.038, n=4, 12h) decreased after LC3 gene; the transcription level was higher than the control group (0.120 + 0.069vs3.104 + 1.191, P=0.00, n=10), decreased. CLP8h and 20h were observed in intestinal epithelial cells, mitochondria swelling, dissolution, endoplasmic reticulum vacuolation, CLP8h intestinal epithelial cells in autophagy and autophagy lysosome number increased significantly than in group 20h (19.33 + 2.16vs9.33+1.86,37.67 + 3.27vs19.50 + 1.87, P=0.00, n=3), CLP20h cells could also be observed in larger autolysosomes.
Conclusion: the sepsis rat small intestine early local and systemic inflammatory cytokines peaked at TNF-a, autophagy related protein LC3 in intestinal mucosa protein and gene expression levels rise in early stage, then decreased by autophagy. Changes of inflammatory cytokines in sepsis associated with pathological conditions, TNF-a water level.
The second part: the role of brain gut peptide Ghrelin in regulating the autophagy of intestinal epithelial cells in septic rats
Objective: To study the regulatory effect of brain gut peptide Ghrelin on intestinal epithelial autophagy in rats with sepsis, and to explore the protective effect of Ghrelin on intestinal tract in rats with sepsis by regulating autophagy.
Methods: 40 male SD rats were randomly divided into normal group, sham operation group, CLP20h group and Ghrelin group, the establishment of animal models for sepsis in rats by cecal ligation and puncture, and the rat tail vein infusion Ghrelin20h, then sacrificed animal, collect serum and intestinal tissue of rats; interception part of the small intestine segment fixation after pathology and electron microscopy, another part of the small intestine at low temperature to scrape the intestinal mucosa for molecular biological detection. The pathological changes were observed in intestinal epithelial optical microscopy, transmission electron microscopy of intestinal epithelial cell structure changes and micro morphology and number of autophagic vacuoles was observed by immunofluorescence to detect the expression of GFP-LC3. The level of.Western-blot and Real-time in intestinal mucosa of autophagy related protein PCR LC3 levels in intestinal epithelial cells, enzyme-linked immunosorbent assay to detect the serum and mucosal inflammation in animal by Zi Shui The difference between each group was analyzed statistically.
Results: (1) compared with the normal group and sham operation group, sepsis 20h rat intestinal epithelial injury obviously, serum and mucosal TNF-a (154.57 + 19.54vs88.58 + 8.19,96.08 + 9.93,74.95 + 4.89vs19.94 + 1.10,22.44 + 4.18, P=0.00, n=10) and MCP-1 (96.33 + 4.89vs61.66 + 6.94,69.16 + 10.42,66.74 + 4.80vs41.74 + 1.78,49.24 + 4.32, P=0.00, n=10) level increased, LC3 protein expression increased (1.52 + 0.03vs0.98 + 0.18,1.25 + 0.09, P=0.00, n=4), the gene transcription level also increased (0.42 + 0.16vs0.13 + 0.11,0.13 + 0.12, P=0.00, n=10). Sepsis intestinal epithelial GFP-LC3 expression in rats with sepsis is higher than the sham operation group (210. 18.03vs168 + 17.66, P=0.003, n=5). The number of autophagic vesicles and intestinal epithelial cell autolysosome is higher than the sham operation group (36.83 + 3.76vs9.33 + 1.86,71.83 + 5.27vs19.50 + 1.87, P=0.00, n=3). (2) compared with the sepsis group, intestinal mucosal capillary congestion Reduce the serum and intestinal mucosa. TNF-a (154.57 + 19.54vs192.75 + 21.91,74.95 + 4.89vs96.85 + 15.35, P=0.00, n=10) and MCP-1 (96.33 + 4.89vs189.93 + 14.59,66.74 + 4.80vs99.08 + 7.47, P=0.00, n=10) decreased, LC3 protein expression increased (1.52 + 0.03vs1.35 + 0.06, P= 0.035, n=4), LC3 (gene transcription 0.42 + 0.16vs0.14 + 0.10, P=0.00, n=10). The increased expression of GFP-LC3 in intestinal epithelial cells (210 + 18.03vs177.80 + 17.66, P=0.014, n=5).Ghrelin group of intestinal epithelial cells in the number of autophagic vacuoles and autophasosomes increased significantly (36.83 + 3.76vs19.33 + 2.16,71.83 + 5.27vs37.67 + 3.27, P=0.00, n=3).
Conclusion: brain gut peptide Ghrelin significantly reduce sepsis in the rat intestinal epithelial injury, reduce the local and systemic inflammatory cytokines, promote intestinal epithelial cell autophagy related protein LC3 expression and transcription, increase the number of autophagic vacuoles with autophagic and lysosomal, intestinal epithelial cells up-regulated in sepsis rats.

【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R459.7

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本文編號(hào)：1435578