脓毒症大鼠小肠上皮自噬水平及脑肠肽Ghrelin对其调节作用的研究

发布时间：2018-01-17 01:00

本文关键词：脓毒症大鼠小肠上皮自噬水平及脑肠肽Ghrelin对其调节作用的研究　出处：《兰州大学》2014年硕士论文　论文类型：学位论文

【摘要】：第一部分：脓毒症大鼠小肠上皮细胞的自噬水平研究目的：研究盲肠结扎穿刺(Cecal ligation and puncture,CLP)诱导的脓毒症大鼠小肠上皮自噬水平的变化,探讨自噬与脓毒症肠上皮损害的关系。方法：雄性SD大鼠60只随机分成对照组,CLP4h组、CLP8h组、CLP12h组、CLP16h组及CLP20h组(n=10)。建立大鼠盲肠结扎穿刺模型后,在各时间点随机处死大鼠,收集大鼠血清及小肠肠段,低温下刮取小肠上皮粘膜保存待检。酶联免疫吸附法检测血清及粘膜细胞因子变化,Western-blot法及Real time-PCR法从蛋白及基因水平检测粘膜组织中自噬相关蛋白LC3表达量,统计学分析各组间差异；透射电子显微镜观察脓毒症8h和20h大鼠肠上皮细胞微细胞结构变化及自噬泡形态,并统计自噬泡及自噬溶酶体数量。结果：CLP诱导的脓毒症大鼠血清及粘膜细胞因子TNF-a和MCP-1在4h即升高,明显高于对照组(P0.05),自噬相关蛋白LC3-Ⅱ/LC3在脓毒症8h明显高于对照组(1.255±0.088vs1.470±0.295,P=0.038,n=4),12h后下降；LC3基因转录水平较对照组升高(0.120±0.069vs3.104±1.191,P=0.00,n=10),随之下降。电镜观察CLP8h和20h肠上皮细胞,线粒体损伤明显,肿胀溶解,内质网空泡化,CLP8h小肠上皮细胞内自噬泡和自噬溶酶体数量较20h组明显增多(19.33±2.16vs9.33+1.86,37.67±3.27vs19.50±1.87,P=0.00,n=3),CLP20h细胞内也可见较大的自噬溶酶体。结论：脓毒症大鼠小肠局部及全身炎性因子TNF-a早期达高峰,小肠粘膜自噬相关蛋白LC3在蛋白和基因水平表达早期升高,随后下降。自噬在脓毒症病理环境下受到炎性细胞因子变化影响,与TNF-a水平正相关。第二部分：脑肠肽Ghrelin对脓毒症大鼠肠上皮细胞自噬的调节作用目的：研究脑肠肽Ghrelin对脓毒症大鼠肠上皮自噬的调节作用,探讨Ghrelin通过调节细胞自噬对脓毒症大鼠肠道发挥保护作用。方法：将40只雄性SD大鼠随机分成正常组、假手术组、CLP20h组及Ghrelin干预组,通过大鼠盲肠结扎穿刺术建立脓毒症动物模型,并经大鼠尾静脉持续泵注Ghrelin20h,随后处死各组动物,收集大鼠血清及小肠组织；截取部分小肠段固定后待行病理及电镜观察,另一部分小肠在低温下刮取肠粘膜待行分子生物学检测。光学显微镜观察各组小肠上皮的病理改变,透射电子显微镜观察小肠上皮微细胞结构变化及细胞内自噬泡形态和数量。免疫荧光观察小肠上皮细胞内GFP-LC3表达水平。Western-blot及Real-time PCR检测小肠粘膜自噬相关蛋白LC3水平,酶联免疫吸附法检测各组动物血清和粘膜内炎症因子水平的变化,统计学分析各组间差异。结果：(1)与正常组及假手术组相比,脓毒症20h大鼠肠上皮病理损伤明显,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蛋白表达升高(1.52±0.03vs0.98±0.18、1.25±0.09,P=0.00,n=4),其基因转录水平也升高(0.42±0.16vs0.13±0.11、0.13±0.12,P=0.00,n=10)。脓毒症大鼠小肠上皮GFP-LC3表达较假手术组升高(210±18.03vs168±17.66,P=0.003,n=5)。小肠上皮细胞自噬泡及自噬溶酶体数量较假手术组升高(36.83±3.76vs9.33±1.86,71.83±5.27vs19.50±1.87,P=0.00,n=3)。(2)与脓毒症组比较,小肠上皮粘膜毛细血管充血减轻。血清及肠粘膜组织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蛋白表达升高(1.52±0.03vs1.35±0.06,P=0.035,n=4),LC3基因转录水平(0.42±0.16vs0.14±0.10,P=0.00,n=10)。肠上皮细胞GFP-LC3表达增多(210±18.03vs177.80±17.66,P=0.014,n=5)。Ghrelin组肠上皮细胞内自噬泡及自噬溶酶体数量明显升高(36.83±3.76vs19.33±2.16,71.83±5.27vs37.67±3.27,P=0.00,n=3)。结论：脑肠肽Ghrelin明显减轻脓毒症大鼠小肠上皮病理损害,降低局部及全身炎症因子水平,促进小肠上皮细胞自噬相关蛋白LC3表达及转录,增加细胞内自噬泡及自噬溶酶体数量,具有上调脓毒症大鼠小肠上皮细胞自噬的作用。
[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.

【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R459.7

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