本文選題：CTCE + miR-126�。� 參考：《吉林大學(xué)》2016年博士論文

【摘要】：敗血癥是指病菌侵入血液循環(huán)中過度繁殖引起的全身性感染,重度敗血癥會導(dǎo)致體內(nèi)炎癥反應(yīng)失調(diào)和多器官功能衰竭。細(xì)菌在血液中大量繁殖并且產(chǎn)生毒素,會破壞肺部血管內(nèi)皮細(xì)胞的功能引起急性肺損傷(Acute Lung Injury,ALI)繼而發(fā)展為死亡率非常高的急性呼吸窘迫綜合癥(Acute Respiratory Distress Syndrome,ARDS),ARDS是重度敗血癥最常見也是最危險的并發(fā)癥。由此可見清除體內(nèi)感染病菌和增強血管內(nèi)皮細(xì)胞屏障功能對提高重度敗血癥患者存活率尤為重要。目前敗血癥的常規(guī)治療主要依靠注射抗生素。然而由于現(xiàn)代社會大量的濫用抗生素,導(dǎo)致了許多耐藥病菌出現(xiàn),加大了敗血癥的治療難度,因此研發(fā)一種可以同時增強機體殺菌能力并能增強血管內(nèi)皮細(xì)胞屏障功能的藥物是現(xiàn)在重度敗血癥治療的研究熱點。CTCE-0214(CTCE)是基質(zhì)細(xì)胞衍生因子-1α(Stromal cell derived factor-1α,SDF-1α)的小肽類分子模擬物,SDF-1α是趨化因子CXC亞家族成員,在體內(nèi)可以激活并募集中性粒細(xì)胞,但SDF-1α在血液中半衰期極短,限制了其應(yīng)用。CTCE在血液循環(huán)中穩(wěn)定性與生物利用度均遠(yuǎn)高于SDF-1α。前期研究結(jié)果表明CTCE可以提高盲腸結(jié)扎穿孔(Cecal ligation and puncture,CLP)所導(dǎo)致實驗性敗血癥小鼠的存活率。本論文旨在研發(fā)一種同時增強機體殺菌能力并能增強血管內(nèi)皮細(xì)胞屏障功能的敗血癥治療藥物。因此通過研究CTCE對敗血癥患者體內(nèi)中性粒細(xì)胞殺菌功能以及對ARDS中肺血管內(nèi)皮細(xì)胞屏障功能的影響可以將為CTCE開發(fā)為新的治療敗血癥的藥物提供理論和實驗依據(jù)。本論文采用如下策略:(1)在體外實驗中測定CTCE對多核中性粒細(xì)胞(Polymorphonuclear neutrophils,PMN)的殺菌能力的影響。(2)在體外實驗中測定CTCE對凝血酶誘導(dǎo)引起的人類肺部微血管內(nèi)皮細(xì)胞(Human lung microvascular endothelial cell,HMVEC)通透性增強的抑制作用并研究其機制。(3)在抗生素治療失效的CLP誘導(dǎo)的重度敗血癥小鼠模型中測定CTCE對小鼠存活率和小鼠體內(nèi)殺菌能力的影響(4)在LPS誘導(dǎo)形成的ARDS小鼠模型中測定CTCE對血管屏障完整性的影響。主要研究方法和結(jié)果如下:(1)通過體外誘導(dǎo)MPRO細(xì)胞分化得到PMN細(xì)胞,測定CTCE對中性粒細(xì)胞體內(nèi)ROS產(chǎn)量和對大腸桿菌K12殺菌能力的影響。結(jié)果顯示CTCE提高PMN細(xì)胞內(nèi)ROS的生成量(增加了31.9±3.7%)以及增強PMN殺菌能力(CFU減少了26.5±3.2%)。由于mi R-223有負(fù)向調(diào)節(jié)PMN細(xì)胞的功能,本論文也對mi R-223的表達量進行了測定。與空白組相比CTCE降低PMN細(xì)胞內(nèi)mi R-223的表達量(降低了38±7.9%)。表明CTCE可以在體外增強PMN細(xì)胞的殺菌功能。(2)體外培養(yǎng)人類肺部微血管內(nèi)皮細(xì)胞(HMVECs),用凝血酶誘導(dǎo)干擾內(nèi)皮細(xì)胞之間的結(jié)構(gòu)完整性,測定CTCE對這種干擾的影響。我們通過測定跨內(nèi)皮細(xì)胞膜電阻來表征內(nèi)皮細(xì)胞屏障功能完整性,同時測定CTCE造成的與內(nèi)皮細(xì)胞屏障完整性相關(guān)的mi RNA表達量和信號通路的變化。結(jié)果顯示CTCE能抑制凝血酶誘導(dǎo)的血管內(nèi)皮細(xì)胞滲漏。同時CTCE還可以恢復(fù)因凝血酶誘導(dǎo)的細(xì)胞表面VE-cadherin的表達量降低以及減少生成的細(xì)胞間隙數(shù)量。CTCE可以提高細(xì)胞內(nèi)mi R-126的表達量并且激活A(yù)KT/Rac 1信號通路,然而用mi R-126抑制劑與CTCE共同處理細(xì)胞時會導(dǎo)致CTCE對AKT的激活被阻止,并且CTCE對血管內(nèi)皮細(xì)胞通透性的保護能力減弱。說明CTCE可以通過提高細(xì)胞內(nèi)mi R-126的表達而激活A(yù)KT/Rac 1信號通路以增強血管內(nèi)皮細(xì)胞結(jié)構(gòu)和功能完整性。(3)將CD-1小鼠分為Sham,Sham+CTCE,CLP和CLP+CTCE四個試驗組,每組十五只,在CLP處理小鼠之后2小時注射亞胺培南(25mg/kg)建立抗生素失效時重癥敗血癥小鼠模型,用CTCE(10mg/kg)或空白生理鹽水在不同時間進行皮下注射。測定7天存活率及小鼠體內(nèi)PMN細(xì)胞殺菌能力。結(jié)果顯示CLP+CTCE組小鼠的血液中PMN的含量相對于Sham組分別在18小時增加到2.4±0.57倍,24小時增加到2.9±0.63倍;同時腹腔液中的PMN含量在24小時增加至CLP對照組的2.0±0.2倍。與CLP組相比,CTCE顯著降低了細(xì)菌的入侵,腹腔液中CFU值減少了78±8.5%,血液中CFU值減少了77±11.4%,肺組織內(nèi)CFU值減少了79±8.3%。CTCE還可以增強PMN的吞噬能力,CTCE對PMN募集能力的增強以及細(xì)菌清除能力的提高使得小鼠死亡率明顯降低,CLP+CTCE組的死亡率從CLP組的67%降低到20%。上述結(jié)果表明CTCE可以通過增加對PMN募集和增強PMN殺菌功能來提高重度敗血癥小鼠的存活率。(4)將CD-1小鼠分為對照組,LPS組和LPS+CTCE三組,每組6-10只,采用氣管內(nèi)滴注LPS來誘導(dǎo)小鼠產(chǎn)生ARDS,之后靜脈注射CTCE。24小時后處死小鼠,提取小鼠肺組織,對血清和支氣管肺泡灌洗液(bronchoalveolar lavage fluid,BAL)進行檢測。結(jié)果顯示靜脈注射CTCE可以顯著減少小鼠支氣管肺泡灌洗液(BAL)中蛋白質(zhì)和免疫細(xì)胞的含量,降低BAL內(nèi)炎癥細(xì)胞因子和趨化因子的含量,并且增加肺組織內(nèi)mi R-126的表達量,減少了肺部血管滲出和肺泡水腫。說明CTCE可以降低LPS誘導(dǎo)小鼠ARDS損傷程度。本論文通過實驗證明在重度敗血癥治療中,CTCE可以通過募集和增強PMN細(xì)胞的殺菌功能降低小鼠的死亡率,在敗血癥并發(fā)癥ARDS中,CTCE可以通過提高mi R-126的表達量和激活A(yù)KT/Rac 1信號通路來保護血管內(nèi)皮細(xì)胞的屏障完整性。CTCE同時具有增強機體殺菌能力和保護血管內(nèi)皮細(xì)胞屏障功能的能力,具有被開發(fā)為治療敗血癥藥物的潛質(zhì)。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R515.3

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