PLK1调节NF-κB通路在脓毒症肠屏障功能障碍中的作用

发布时间：2018-04-08 22:04

  本文选题：脓毒症　切入点：肠屏障　出处：《皖南医学院》2015年硕士论文

【摘要】：目的通过在体实验和离体实验,探讨PLK1和NF-?B信号通路在脓毒症肠屏障功能障碍中的作用。方法在体实验:取健康清洁成年雄性C57BL小鼠40只,采用随机数字法分为2组(n=20):正常对照组(C组)和脓毒症组(LPS组),采用腹腔内注射脂多糖(LPS)40mg/kg制备脓毒症小鼠模型,对照组腹腔注射等量生理盐水。造模12h后,取小鼠静脉血,测定血清TNF-?及二胺氧化酶(DAO)水平,然后用颈椎脱臼法处死,取回盲部小肠5cm.,4%多聚甲醛固定,常规脱水石蜡包埋,切片,HE染色,观察小肠绒毛与黏膜炎症等病理改变。采用TUNEL法进行原位细胞凋亡检测。小鼠小肠PLK1表达水平检测采用Western blotting及免疫组织化学的方法。离体实验:在HT29细胞培养基中加入不同浓度(0,10,20,30?g/ml)的LPS处理24h,收集细胞行Annexin V/Propidium Iodide染色后用流式细胞分析仪检测细胞凋亡情况,同时提取蛋白行Western blotting检测PARP1、Caspase3、PLK1、I?B-?等蛋白表达水平,并采用细胞免疫荧光法检测NF-?B蛋白定位;在HT29细胞培养基中加入不同浓度(0,2,10,20?ng/ml)的NF-?B通路抑制剂PDTC预处理4h,再加入LPS(30?g/ml)处理24h,收集细胞行Annexin V/Propidium Iodide染色后用流式细胞分析仪检测细胞凋亡情况,同时提取蛋白行Western blotting检测Caspase3、I?B-?等蛋白表达水平;在HT29细胞培养基中加入PLK1抑制剂BI2536(50nM)处理24h,收集细胞提取蛋白行Western blotting检测PARP1、PLK1、I?B-?等蛋白表达水平,并采用细胞免疫荧光法检测NF-?B蛋白定位;在HT29细胞中正义转染pCDNA3.1-PLK1-myc质粒,再加入LPS(30?g/ml)处理24h,收集细胞用Annexin V/Propidium Iodide染色后行流式细胞分析仪检测细胞凋亡情况,同时提取蛋白行Western blotting检测PARP1、Caspase3等蛋白表达水平。结果在体实验:(1)腹腔注射LPS(40mg/kg)后,与C组相比,LPS组小鼠精神萎靡、动作迟缓,腹腔内有大量脓性积液,肠管充血水肿,血清TNF-?浓度显著增加(P0.001)。(2)与C组相比,LPS组小鼠肠黏膜明显萎缩,小肠绒毛脱落,腺体结构丧失,血清DAO浓度显著增加(P0.001)。(3)与C组相比,LPS组小鼠肠黏膜上皮细胞发生凋亡,小肠组织中PARP1,Caspase3蛋白表达水平明显下调。(4)与C组相比,LPS组小鼠小肠组织中PLK1表达减少,I?B-?蛋白的表达水平下调。离体实验:(1)不同浓度LPS处理HT29细胞24h,可诱导细胞发生凋亡,且凋亡细胞比例随LPS浓度的增加而增大。(2)不同浓度LPS处理HT29细胞24h,与对照组相比,细胞核中NF-?B蛋白表达增加,同时I?B-?蛋白的表达水平下调。(3)用PDTC(10 ng/ml,20?ng/ml)预处理HT29细胞4h,与对照组相比,可显著减少LPS引起的HT29细胞凋亡比例(P0.01或P0.001)。(4)不同浓度LPS处理HT29细胞24h,与对照组相比,PLK1蛋白的表达水平下调。(5)BI2536(50nM)处理HT29细胞24h,与对照组相比,细胞核中NF-?B蛋白表达增加,同时I?B-?、PARP1蛋白的表达水平下调。(6)在HT29细胞中正义转染pCDNA3.1-PLK1-myc质粒,与未正义转染组细胞相比,LPS(30?g/ml)处理诱导细胞产生的凋亡比例显著减少(P0.01)。结论PLK1蛋白表达下调和NF-?B通路激活在脓毒症肠黏膜屏障障碍机制中发挥重要作用;脓毒症时,PLK1通过负向调节NF-?B通路,加重肠上皮细胞凋亡,引起肠黏膜通透性增加,导致肠屏障功能障碍。
[Abstract]:Objective by in vivo and in vitro experiments, to investigate the PLK1 and NF-? B signal pathway in sepsis with intestinal barrier dysfunction in in vivo. Methods: healthy clean adult male C57BL 40 mice were randomly divided into 2 groups (n=20): normal control group (C group) and sepsis group (LPS group), by intraperitoneal injection of lipopolysaccharide (LPS) sepsis mice model of 40mg/kg prepared by the control group, intraperitoneal injection of saline. 12h after modeling, the mice blood serum TNF-? Two and monoamine oxidase (DAO), and then were executed by cervical dislocation. Ileocecal intestinal 5cm., 4% paraformaldehyde, conventional dehydration, paraffin embedding, slice, HE staining, and the intestinal villi mucosa inflammation and other pathological changes were observed by TUNEL assay. Cell apoptosis in mouse intestinal. The expression level of PLK1 detected by Western blotting and immunohistochemistry. In vitro experiments: in the medium added with the different concentrations of HT29 cell culture (0,10,20,30? G/ml) LPS 24h, Annexin V/Propidium cells were collected after Iodide staining by flow cytometry and cell apoptosis, simultaneous extraction of protein by Western blotting detection of PARP1, Caspase3, PLK1, I? B-? Protein expression level, and by immunofluorescence detection of NF-? B protein; in the medium added with the different concentrations of HT29 cell culture (0,2,10,20? Ng/ml) NF-? B pathway inhibitor PDTC pretreated 4h, adding LPS (30? G/ml) 24h, Annexin V/Propidium cells were collected after Iodide staining by flow cytometry and cell apoptosis at the same time, Western protein extraction blotting detection Caspase3, I? B-? Protein expression level; in addition of the PLK1 inhibitor BI2536 based in HT29 cell culture (50nM) at 24h, Western blotting cells were collected to extract protein The detection of PARP1, PLK1, I? B-? Protein expression level, and the immunofluorescence detection of NF-? B protein in HT29 cells; justice transfected with pCDNA3.1-PLK1-myc plasmid, then add LPS (30? G/ml) 24h, flow cytometry and apoptosis cells were collected with Annexin V/Propidium staining for Iodide at the same time, Western blotting detection of PARP1 protein extraction, the expression level of Caspase3 protein. Results: (1) intraperitoneal injection of LPS (40mg/kg), compared with the C group, LPS group, apathetic, slow, there is a lot of purulent fluid of abdominal cavity, bowel edema, serum TNF- concentration increased significantly? (P0.001). (2) compared with C group, LPS group of mice intestinal mucosal atrophy, intestinal villus shedding, glandular structure loss, the concentration of serum DAO increased significantly (P0.001). (3) compared with the C group, LPS group of mice intestinal mucosal epithelial cell apoptosis of intestinal tissue 涓璓ARP1,Caspase3铔嬬櫧琛ㄨ揪姘村钩鏄庢樉涓嬭皟.(4)涓嶤缁勭浉姣，

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