诱导型多能干细胞通过修复ZO-1蛋白改善脓毒症小鼠肠黏膜通透性和细菌移位

发布时间：2017-12-31 14:01

本文关键词：诱导型多能干细胞通过修复ZO-1蛋白改善脓毒症小鼠肠黏膜通透性和细菌移位　出处：《中山大学学报(医学科学版)》2016年05期 　论文类型：期刊论文

【摘要】：【目的】探讨诱导型多能干细胞(i PS-MSC)对脓毒症小鼠肠黏膜通透性的改善作用和对肠道菌群移位的影响。【方法】采用尾静脉注射脂多糖(LPS,10 mg/Kg)建立小鼠脓毒症模型。常规培养及传代i PS-MSC。在脓毒症建立2 h和6 h经小鼠尾静脉注射进行治疗干预(1×106/只)。利用基因重组技术制备新质粒p ET28b(+)-EGFP,转染大肠杆菌,并在脓毒症后连续2 d予小鼠进行灌胃处理。在脓毒症发生72 h后,取各组小鼠腹水及肠系膜淋巴结匀浆于Kan抗性的LB培养基培养鉴定,计算和比较菌群负荷。通过透射电镜观察小鼠小肠黏膜上皮细胞超微结构的变化。Western blotting法检测各组小鼠肠黏膜ZO-1蛋白的表达。荧光显微镜下观察带有GFP绿色荧光蛋白的ips-MSC在损伤肠黏膜的移行和归巢情况。【结果】成功构建表达绿色荧光的重组大肠杆菌p ET-28b(+)-EGFP作为实验用示踪菌。灌胃处理后,ips-MSC治疗的小鼠腹水和肠系膜淋巴结的示踪菌的菌负荷较脓毒症组明显降低,其中ips-MSC 2 h治疗组较6 h治疗组降低的更明显。透射电镜观察发现脓毒症小鼠肠系膜上皮细胞间紧密连接结构破坏,细胞间缝隙增宽,而经i PS-MSC治疗的小鼠细胞间紧密连接结构破坏明显改善,在2 h治疗组表现更为明显,伴随紧密连接蛋白ZO-1的表达水平升高。【结论】静脉注射i PS-MSC能够向脓毒症小鼠损伤的肠黏膜移行,通过提高连接蛋白ZO-1的表达,修复紧密连接,改善脓毒症小鼠肠黏膜的通透性,减少脓毒症时肠道菌群的移位。
[Abstract]:[Objective] to investigate the induced pluripotent stem cells (I PS-MSC) to improve the role of sepsis in colonic mucosa of mice with sepsis and the influence on the permeability of bacterial translocation. [method] by intravenous injection of lipopolysaccharide (LPS, 10 mg/Kg) to establish a mouse model of sepsis. The conventional culture and subculture I PS-MSC. established in sepsis in 2 h and 6 h were injected into tail vein of mice were treated (1 x 106/). Preparation of plasmid P ET28b by gene recombination technology (+) -EGFP, transfected into Escherichia coli, and 2 consecutive d to mice in sepsis after gavage. In sepsis after 72 h from each group, the ascites and mesenteric lymph nodes of mice in homogenate Kan resistant LB medium identification, calculation and comparison of bacteria load. Transmission electron microscope was used to observe changes of.Western expression by blotting mice intestinal mucosal epithelial cell ultrastructure of intestinal mucosa of mice were detected ZO-1 protein fluorescence. Light microscope with GFP green fluorescent protein ips-MSC in intestinal mucosa injury of migration and homing. [results] the expression of green fluorescence in recombinant Escherichia coli P was successfully constructed ET-28b (+) -EGFP as tracer experiments with bacteria. After treatment by gavage, bacterial loads in ips-MSC treated mice ascites and mesenteric lymph node tracer bacteria than sepsis group significantly decreased, the ips-MSC 2 h treatment group compared with 6 h treatment group decreased more obvious. Transmission electron microscopy showed sepsis mouse mesenteric tight junctions between epithelial cells damage, cell gap widened, and the cell I of mice treated with PS-MSC significantly improved the close connection between the structural damage in 2, h in treatment group was more obvious, with increased expression of tight junction protein ZO-1. [Conclusion] the intravenous injection of I PS-MSC to septic mice intestinal mucosa injury by increasing the migration The expression of protein ZO-1, repair close connection, improve the permeability of intestinal mucosa in sepsis mice, and reduce the displacement of intestinal microflora in sepsis.

【作者单位】：中山大学附属第一医院急诊科;中山大学附属第三医院急诊科;
【基金】：广东省医学科学技术研究基金(A2015486) 广东省科技计划项目(2012B031800366) 广东省自然科学基金(S2013010015328)
【分类号】：R459.7
【正文快照】： 脓毒症是常并发于创伤、手术、感染、休克等,是目前临床常见的危急重症之一,死亡率高,救治困难[1]。脓毒症的应激状态下,患者的肠黏膜的结构和功能极易受到损伤,表现为肠黏膜萎缩,肠道的通透性增高,肠腔内细菌移位,从而诱发肠原性感染的二次打击,进一步诱发全身炎症反应综合征

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