【作者】 杨涛；

【导师】 于泳浩；

【作者基本信息】 天津医科大学 ， 麻醉学， 2016， 硕士

【摘要】 目的:脓毒症是由感染因素诱发并且以炎性反应为主的综合征,是烧伤、创伤和大手术等的常见并发症。肠上皮屏障能够阻挡微生物、毒素等透过肠壁进入人体内其他组织、器官以及血液循环。肠屏障功能障碍是脓毒症引发多器官功能障碍综合征(MODS)的重要因素。紧密连接蛋白occludin与黏附连接蛋白E-cadherin是肠上皮屏障的主要组成成分。内毒素脂多糖(LPS)通过改变紧密连接蛋白和黏附连接蛋白的正常表达,造成肠上皮屏障通透性增加,引发肠屏障功能障碍。多项研究已经证实氢气对多种脓毒症动物模型具有明确的保护作用,但是其具体机制尚未明确。本实验研究拟运用人结肠腺癌细胞系建立离体人肠屏障功能障碍模型,观察富氢培养液在调节LPS诱发的肠屏障功能障碍中的作用,并深入探讨其相关的作用机制。方法:人结肠腺癌细胞系Caco2复苏后传代培养至第28-35代之间时用于实验。第一部分:建立Caco2细胞单层模型。首先分别采用不同浓度的LPS(1,10,100μg/ml和1 mg/ml)孵育,于不同时间点(0,3,6,12,24,48 h)检测跨上皮电阻(TER)值和异硫氰酸荧光素标记的葡聚糖的渗透系数(PE)。采用CCK-8法测定100μg/ml和1 mg/ml的LPS对Caco2细胞增殖活力的影响。随机将细胞进行分组(4组):对照(control)组、富氢培养液(H2)组、脂多糖(LPS)组和LPS+H2组。按分组处理,于孵24 h时,分别检测TER值和PE。分别于孵育6、12和24 h时,采用Western blot法测定occludin与E-cadherin的表达水平;于孵育24 h时,采用免疫荧光法测定occludin与E-cadherin的分布情况。第二部分:首先按照第一部分的分组方法于孵育24h时,采用GST-Pulldown法测定Caco2细胞中Ras同源家族蛋白A(Rho A)的活性。细胞进行随机分组(5组):control组、LPS组、LPS+H2组、LPS+H 2+Rho A激动剂CN03(LPS+H2+CN03)组、LPS+Rho A抑制剂C3胞外酶(LPS+C3)组。Control组、LPS组以及LPS+H2组处理同第一部分。LPS+H2+CN03组中,于孵育前3 h时加入1μg/ml的CN03。LPS+C3组中,于孵育前1 h时加入2.5μg/ml的C3胞外酶。于孵育24 h时测定肠屏障模型的TER值与PE。分别采用Western blot法和免疫荧光法测定occludin与E-cadherin的表达与分布情况。第三部分:首先按照第一部分的分组方法于孵育24h时,Western blot法测定哺乳动物Diaphanous相关成蛋白1(m Dia1)的表达水平。慢病毒法转染Caco2细胞,对m Dia1进行RNA干扰,并获取稳定转染细胞株。将细胞随机分为5组:control组、LPS组、RNA干扰(si RNA m Dia1)组、LPS+H2组和si RNA m Dia1+LPS+H2组。其中,control组、LPS组和LPS+H2组处理同前;si RNA m Dia1组和si RNA m Dia1+LPS+H2组使用稳定表达m Dia1干扰片段的Caco2细胞。于孵育24 h时测定肠屏障模型的TER值与PE。分别采用Western blot法和免疫荧光法测定occludin与E-cadherin的表达与分布情况。结果:第一部分:与0μg/ml LPS组比较,100μg/ml和1 mg/ml的LPS孵育6-48h的时间范围内,TER值下降且PE均降低升高,趋势呈现出时间依赖性,并且在24 h时变化程度最为明显;100μg/ml LPS对细胞活力无显著影响,而1mg/ml LPS组中细胞活力显著降低。Control组与H2组比较,上述各指标间差异均无统计学意义。与control组比较,在LPS组孵育24 h时肠屏障模型的TER值降低,PE升高;孵育6-24 h时,occludin和E-cadherin的表达均下调;孵育24 h时,occludin和E-cadherin在细胞膜处分布减少,在细胞质中分布增多,“铁丝网”样形态的连续性与完整性破坏。与LPS组比较,LPS+H2组孵育24 h时肠屏障模型的TER值升高,PE降低;孵育6-24 h时,occludin和E-cadherin的表达均上调;孵育24 h时,occludin和E-cadherin在细胞膜处分布增多,在细胞质中分布减少,“铁丝网”样形态的连续性与完整性改善。第二部分:与control组比较,LPS组中Rho A活性升高;与LPS组比较,LPS+H2组中Rho A活性降低。与LPS+H2组比较,LPS+H2+CN03组中TER值降低且PE升高,occludin和E-cadherin表达下调,且分布紊乱。与LPS组比较,LPS+C3组中,TER值升高且PE降低,occludin和E-cadherin的表达上调,分布情况改善。第三部分:与control组比较,LPS组m Dia1表达降低,LPS+H2组m Dia1表达升高。与control组比较,si RNA m Dia1组TER值降低且PE升高,occludin和E-cadherin表达下调,且分布紊乱;与LPS+H2组比较,si RNA m Dia1+LPS+H2组TER值降低且PE升高,occludin和E-cadherin表达下调,且分布紊乱。结论:富氢培养液能够改善LPS所致离体人肠屏障模型的通透性异常增加,并能调节紧密连接蛋白occludin和黏附连接蛋白E-cadherin的表达与分布,体现了肠屏障保护效应,其机制可能与调节Rho A-m Dia1通路的表达水平有关。更多还原

【Abstract】 Objective: Sepsis is characterized by a severe inflammatory response to infection. The gastrointestinal epithelium forms the boundary between the body and external environment and provides a selective permeable barrier achieved by tight junction(TJ) and adherens junction(AJ) structures. Recently, H2 has been demonstrated to effectively ameliorate intestinal injury and MODS in septic mice. The purpose of this study aimed at researching the effects of molecular hydrogen on endotoxin-induced intestinal barrier dysfunction in vitro and the mechanisms involved.Methods: Caco2 cells were cultured to passage 28-35 in culture medium. Part 1: To establish intestinal epithelial models in vitro. At first, cells were treated with LPS in different concentrations(1, 10,100 μg/ml and 1 mg/ml) for different time points(0, 3, 6, 12, 24, 48 h) to measure the transepithelial electrical resistance(TER) and FITC-dextran flux(PE). Then Caco2 cells treated 100 μg/ml or 1 mg/ml LPS were measured by CCK-8 assay to detect cell viability. After these, Cells were divided into 4 groups: control group, hydrogen-rich medium(H2) group, LPS group and LPS + H2 group. Cells were cultured in hydrogen-rich medium in H2 group and LPS + H2 group. Meanwhile, 100 μg/ml LPS was simultaneously added into LPS group and LPS + H2 group. TER and PE from each group were measured at 24 h; The expression levels of occludin and E-cadherin were respectively detected at 6, 12 and 24 h Western blot analysis; The morphological structures of occludin and E-cadherin were observed at 24 h by immunofluorescence staining. Part 2: The levels of activated Ras homolog family member A(Rho A) in each group of Part 1 were measured at 24 h by GST pull-down assay. Cells were randomly divided into 5 groups: control group, LPS group, LPS + H2 group, LPS + H2 + CN03 group and LPS + C3 exoenzyme(LPS + C3) group. The treatment methods of control group, LPS group and LPS + H2 group were in line with Experiment One. 2.5μg/ml C3 exoenzyme was added to LPS + C3 group 1 h before incubation, 1 μg/ml CN03 added to LPS + H2 + CN03 group 3 h before incubation. TER and PE as well as expressions and structures of occludin and E-cadherin in each group were measured.Part 3: Expressions of Mammalian diaphanous-related formin1(m Dia1) in each group of Part 1 were detected at 24 h. Caco2 were transfected to establish si RNA m Dia1 mutant. Cells were divided into 5 groups: control group, LPS group, si RNA m Dia1 group, LPS + H2 group, si RNA m Dia1 + LPS + H2 group. The treatment of control group, LPS group, LPS+H2 group were in line with Part 1. TER, PE and expressions and structures of occludin and E-cadherin in each group were measured.Results: Part 1: 100 μg/ml and 1 mg/ml LPS changed TER and PE. 100 μg/ml LPS caused no cytotoxicity. Compared with control group, LPS group showed that besides the increased PE, others were all decreased; the structures of occludin and E-cadherin were disrupted. Compared with LPS group, LPS + H2 group showed that, besides the decreased PE, others were all dropped. Part 2: LPS activated Rho A and H2 lightened its activity. Compared with LPS group, LPS + H2 group and LPS + C3 group showed that, besides the decreased PE, others were all dropped; the disrupted structures of occludin and E-cadherin were partly reversed. Compared with LPS + H2 group, LPS + H2 + CN03 group showed that, besides the increased PE, others were all decreased; the structures of occludin and E-cadherin were disrupted. Part 3: LPS upregulated m Dia1 and H2 downregulated m Dia1. Compared with control group, LPS group and si RNA m Dia1 group showed that, besides the increased PE, others were all decreased; the structures of occludin and E-cadherin were disrupted. Compared with LPS + H2 group, LPS + H2 + CN03 group showed that, besides the increased PE, others were all decreased; the structures of occludin and E-cadherin were disrupted.Conclusion: Hydrogen-rich medium can attenuate LPS-causedgut barrier dysfunction in vitro via regulating Rho A-m Dia1 signaling, thereby lightening the down-regulated expressions and reversing disrupted structure changes of occludin and E-cadherin.更多还原