SDF-1α模拟物CTCE-0214对败血症的治疗作用研究

发布时间：2018-06-23 09:17

本文选题：CTCE + miR-126　；参考：《吉林大学》2016年博士论文

【摘要】：败血症是指病菌侵入血液循环中过度繁殖引起的全身性感染,重度败血症会导致体内炎症反应失调和多器官功能衰竭。细菌在血液中大量繁殖并且产生毒素,会破坏肺部血管内皮细胞的功能引起急性肺损伤(Acute Lung Injury,ALI)继而发展为死亡率非常高的急性呼吸窘迫综合症(Acute Respiratory Distress Syndrome,ARDS),ARDS是重度败血症最常见也是最危险的并发症。由此可见清除体内感染病菌和增强血管内皮细胞屏障功能对提高重度败血症患者存活率尤为重要。目前败血症的常规治疗主要依靠注射抗生素。然而由于现代社会大量的滥用抗生素,导致了许多耐药病菌出现,加大了败血症的治疗难度,因此研发一种可以同时增强机体杀菌能力并能增强血管内皮细胞屏障功能的药物是现在重度败血症治疗的研究热点。CTCE-0214(CTCE)是基质细胞衍生因子-1α(Stromal cell derived factor-1α,SDF-1α)的小肽类分子模拟物,SDF-1α是趋化因子CXC亚家族成员,在体内可以激活并募集中性粒细胞,但SDF-1α在血液中半衰期极短,限制了其应用。CTCE在血液循环中稳定性与生物利用度均远高于SDF-1α。前期研究结果表明CTCE可以提高盲肠结扎穿孔(Cecal ligation and puncture,CLP)所导致实验性败血症小鼠的存活率。本论文旨在研发一种同时增强机体杀菌能力并能增强血管内皮细胞屏障功能的败血症治疗药物。因此通过研究CTCE对败血症患者体内中性粒细胞杀菌功能以及对ARDS中肺血管内皮细胞屏障功能的影响可以将为CTCE开发为新的治疗败血症的药物提供理论和实验依据。本论文采用如下策略:(1)在体外实验中测定CTCE对多核中性粒细胞(Polymorphonuclear neutrophils,PMN)的杀菌能力的影响。(2)在体外实验中测定CTCE对凝血酶诱导引起的人类肺部微血管内皮细胞(Human lung microvascular endothelial cell,HMVEC)通透性增强的抑制作用并研究其机制。(3)在抗生素治疗失效的CLP诱导的重度败血症小鼠模型中测定CTCE对小鼠存活率和小鼠体内杀菌能力的影响(4)在LPS诱导形成的ARDS小鼠模型中测定CTCE对血管屏障完整性的影响。主要研究方法和结果如下:(1)通过体外诱导MPRO细胞分化得到PMN细胞,测定CTCE对中性粒细胞体内ROS产量和对大肠杆菌K12杀菌能力的影响。结果显示CTCE提高PMN细胞内ROS的生成量(增加了31.9±3.7%)以及增强PMN杀菌能力(CFU减少了26.5±3.2%)。由于mi R-223有负向调节PMN细胞的功能,本论文也对mi R-223的表达量进行了测定。与空白组相比CTCE降低PMN细胞内mi R-223的表达量(降低了38±7.9%)。表明CTCE可以在体外增强PMN细胞的杀菌功能。(2)体外培养人类肺部微血管内皮细胞(HMVECs),用凝血酶诱导干扰内皮细胞之间的结构完整性,测定CTCE对这种干扰的影响。我们通过测定跨内皮细胞膜电阻来表征内皮细胞屏障功能完整性,同时测定CTCE造成的与内皮细胞屏障完整性相关的mi RNA表达量和信号通路的变化。结果显示CTCE能抑制凝血酶诱导的血管内皮细胞渗漏。同时CTCE还可以恢复因凝血酶诱导的细胞表面VE-cadherin的表达量降低以及减少生成的细胞间隙数量。CTCE可以提高细胞内mi R-126的表达量并且激活AKT/Rac 1信号通路,然而用mi R-126抑制剂与CTCE共同处理细胞时会导致CTCE对AKT的激活被阻止,并且CTCE对血管内皮细胞通透性的保护能力减弱。说明CTCE可以通过提高细胞内mi R-126的表达而激活AKT/Rac 1信号通路以增强血管内皮细胞结构和功能完整性。(3)将CD-1小鼠分为Sham,Sham+CTCE,CLP和CLP+CTCE四个试验组,每组十五只,在CLP处理小鼠之后2小时注射亚胺培南(25mg/kg)建立抗生素失效时重症败血症小鼠模型,用CTCE(10mg/kg)或空白生理盐水在不同时间进行皮下注射。测定7天存活率及小鼠体内PMN细胞杀菌能力。结果显示CLP+CTCE组小鼠的血液中PMN的含量相对于Sham组分别在18小时增加到2.4±0.57倍,24小时增加到2.9±0.63倍;同时腹腔液中的PMN含量在24小时增加至CLP对照组的2.0±0.2倍。与CLP组相比,CTCE显著降低了细菌的入侵,腹腔液中CFU值减少了78±8.5%,血液中CFU值减少了77±11.4%,肺组织内CFU值减少了79±8.3%。CTCE还可以增强PMN的吞噬能力,CTCE对PMN募集能力的增强以及细菌清除能力的提高使得小鼠死亡率明显降低,CLP+CTCE组的死亡率从CLP组的67%降低到20%。上述结果表明CTCE可以通过增加对PMN募集和增强PMN杀菌功能来提高重度败血症小鼠的存活率。(4)将CD-1小鼠分为对照组,LPS组和LPS+CTCE三组,每组6-10只,采用气管内滴注LPS来诱导小鼠产生ARDS,之后静脉注射CTCE。24小时后处死小鼠,提取小鼠肺组织,对血清和支气管肺泡灌洗液(bronchoalveolar lavage fluid,BAL)进行检测。结果显示静脉注射CTCE可以显著减少小鼠支气管肺泡灌洗液(BAL)中蛋白质和免疫细胞的含量,降低BAL内炎症细胞因子和趋化因子的含量,并且增加肺组织内mi R-126的表达量,减少了肺部血管渗出和肺泡水肿。说明CTCE可以降低LPS诱导小鼠ARDS损伤程度。本论文通过实验证明在重度败血症治疗中,CTCE可以通过募集和增强PMN细胞的杀菌功能降低小鼠的死亡率,在败血症并发症ARDS中,CTCE可以通过提高mi R-126的表达量和激活AKT/Rac 1信号通路来保护血管内皮细胞的屏障完整性。CTCE同时具有增强机体杀菌能力和保护血管内皮细胞屏障功能的能力,具有被开发为治疗败血症药物的潜质。
[Abstract]:Septicaemia is a systemic infection caused by Overpropagation of the pathogenic bacteria in the circulation of blood. Severe septicemia can lead to disorders of inflammation and multiple organ failure in the body. Bacteria propagate in the blood and produce toxins, which will destroy the function of the pulmonary vascular endothelial cells and cause acute lung injury (Acute Lung Injury, ALI). For acute respiratory distress syndrome (Acute Respiratory Distress Syndrome, ARDS), ARDS is the most common and most dangerous complication of severe sepsis. It can be seen that it is particularly important to clear the infection of the body and enhance the barrier function of the vascular endothelial cells to improve the survival rate of patients with severe abortion. Conventional treatment is mainly dependent on antibiotics. However, due to a large number of antibiotics in modern society, many drug resistant pathogens are occurring and the difficulty of treating septicemia is increased. Therefore, the research and development of a drug that can simultaneously enhance the body's bactericidal ability and enhance the vascular endothelial cell barrier function is the Research of severe sepsis. The hot spot.CTCE-0214 (CTCE) is a peptide molecule mimic of matrix cell derived factor -1 alpha (Stromal cell derived factor-1 alpha, SDF-1 a). SDF-1 alpha is a member of the chemokine CXC subfamily, which can activate and recruit granulocytes in the body. But SDF-1 alpha is very short in the blood, which restricts its application to the stability of the blood circulation. Both sex and bioavailability are far higher than SDF-1 alpha. Previous studies showed that CTCE could improve the survival rate of mice with experimental septicemia caused by the cecum ligation perforation (Cecal ligation and puncture, CLP). The purpose of this paper is to develop a septicaemia treatment drug that simultaneously enhances the body's bactericidal ability and enhances vascular endothelial cell barrier function. Therefore, the study of the bactericidal function of neutrophils in septicemia patients and the effect of CTCE on the barrier function of pulmonary vascular endothelial cells in ARDS can provide theoretical and experimental basis for the development of CTCE as a new drug for the treatment of septicemia. The following strategies are adopted in this paper: (1) the determination of CTCE to polymorphonuclear neutrophils in vitro The effect of Polymorphonuclear neutrophils (PMN) on the bactericidal ability. (2) the inhibitory effect of CTCE on the enhancement of the permeability of the human pulmonary microvascular endothelial cells (Human lung microvascular endothelial cell, HMVEC) induced by thrombin in vitro and the mechanism of its mechanism. (3) the CLP induced weight of antibiotic treatment failure The effect of CTCE on the survival rate of mice and the bactericidal ability of mice in mice (4) the effect of CTCE on the integrity of vascular barrier in the ARDS mice induced by LPS. The main research methods and results were as follows: (1) PMN cells were obtained by inducing MPRO cells to differentiate in vitro, and CTCE was determined in neutrophils. ROS yield and effect on bactericidal ability of Escherichia coli K12. The results showed that CTCE increased the production of ROS in PMN cells (increased by 31.9 + 3.7%) and enhanced PMN bactericidal ability (CFU decreased by 26.5 + 3.2%). As mi R-223 had the function of negative regulation PMN cells, the expression of MI R-223 was measured in this paper. The expression of MI R-223 in low PMN cells (decreased by 38 + 7.9%). Indicated that CTCE could enhance the bactericidal function of PMN cells in vitro. (2) human pulmonary microvascular endothelial cells (HMVECs) were cultured in vitro, the structural integrity between endothelial cells was induced by thrombin, and the influence of CTCE on this interference was measured. We measured cross endothelial cells through the determination of cross endothelial cells. The membrane resistance was used to characterize the endothelial barrier function integrity, and the changes in the expression of MI RNA and signal pathways associated with the endothelial barrier integrality were measured by CTCE. The results showed that CTCE could inhibit the leakage of thrombin induced vascular endothelial cells. Meanwhile, CTCE could also restore the surface VE-cadherin induced by thrombin. The reduction in quantity and the decrease of the number of intercellular spaces generated by.CTCE can increase the expression of MI R-126 in cells and activate the AKT/Rac 1 signaling pathway. However, the activation of CTCE against AKT is prevented when the MI R-126 inhibitor and CTCE are treated together with CTCE, and CTCE protects the vascular endothelial cell permeability. The AKT/Rac 1 signal pathway was activated by increasing the expression of intracellular mi R-126 to enhance the structural and functional integrity of vascular endothelial cells. (3) the CD-1 mice were divided into four experimental groups, Sham, Sham+CTCE, CLP and CLP+CTCE, and fifteen mice in each group were injected with imipenem (25mg/kg) to establish antibiotic failure 2 hours after CLP treatment. CTCE (10mg/kg) or blank physiological saline was injected subcutaneously at different time. The 7 day survival rate and the bactericidal ability of PMN cells in mice were measured. The results showed that the content of PMN in the blood of group CLP+CTCE was increased to 2.4 + 0.57 times compared with that of the Sham group, and increased to 2.9 + 0.63 times in 24 hours, and the peritoneal fluid was at the same time. In the 24 hours, the content of PMN increased to 2 + 0.2 times as much as that of the CLP control group. Compared with the CLP group, CTCE significantly reduced the invasion of bacteria, decreased the CFU value in the peritoneal fluid by 78 + 8.5%, the CFU value in the blood decreased by 77 + 11.4%, the CFU value in the lung tissue decreased by 79 + 8.3%.CTCE and could also enhance the swallowing ability of PMN, CTCE to PMN recruitment and refinement. The increase of bacterial scavenging ability significantly reduced the mortality of mice. The mortality of CLP+CTCE group decreased from 67% in group CLP to 20%.. The results showed that CTCE could increase the survival rate of severe septicemia mice by increasing PMN recruitment and enhancing PMN bactericidal function. (4) CD-1 mice were divided into control group, LPS group and LPS+CTCE three groups, 6-10 rats in each group. The mice were induced to produce ARDS by intratracheal infusion of LPS, and then mice were killed after intravenous injection of CTCE.24 hours. The mice lung tissue was extracted and the serum and bronchoalveolar lavage fluid (bronchoalveolar lavage fluid, BAL) were detected. The results showed that intravenous CTCE could significantly reduce the protein and protein in the bronchoalveolar lavage fluid (BAL) of mice. The content of immune cells, reducing the content of inflammatory cytokines and chemokines in BAL, and increasing the expression of MI R-126 in lung tissue, reducing pulmonary vascular exudation and alveolar edema. It shows that CTCE can reduce the degree of ARDS injury induced by LPS in mice. This paper has proved that in the treatment of severe sepsis, CTCE can be raised by recruitment and in the treatment of severe sepsis. Enhancing the bactericidal function of PMN cells reduces the mortality of mice, and in the septicemia complication ARDS, CTCE can protect the barrier integrity.CTCE of vascular endothelial cells by increasing the expression of MI R-126 and activating the AKT/Rac 1 signaling pathway, and it has the ability to enhance the body's bactericidal ability and protect the vascular endothelial cell barrier function. It has been developed as a potential drug for the treatment of septicemia.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R515.3

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