ARDS中LBP对fractalkine表达的影响及信号转导机制

发布时间：2018-07-05 06:39

本文选题：急性呼吸窘迫综合征 + 脂多糖结合蛋白　；参考：《暨南大学》2017年博士论文

【摘要】：研究背景:急性呼吸窘迫综合征(Acute respiratory distress syndrome,ARDS)是临床上常见的危重症,病死率高,内毒素血症介导的炎性反应是ARDS发病的常见原因之一。脂多糖(lipopolysaccharide,LPS)是内毒素的主要成份。脂多糖结合蛋白(lipopolysaccharide binding protein,LBP)是存在于正常人和动物血清中的一种糖蛋白,在炎症反应的急性期升高,参与多种炎症因子的调控。LPS与LBP结合形成的LPS-LBP复合物激活单核细胞、巨噬细胞和其它细胞上的CD14/TLR4受体,Toll样受体4(Toll-like receptor4,TLR4)激活后可介导LPS胞内信号转导,转导通路向两个方向进行,即丝裂原活化蛋白激酶(mitogen activated protein kinase,MAPK)通路和核因子-κB(nuclear factor-κB,NF-κB)通路。MAPK、NF-κB信号通路活化后通过调节下游炎症因子的表达诱发炎症反应。Fractalkine(FKN)是趋化因子CX3C家族的唯一成员,一方面FKN于炎性细胞在血管壁上的募集和内皮细胞的损伤具有重要的作用,而另一方其通过对细胞凋亡的抑制作用起到抗炎作用。目前LBP对LPS激活MAPK、NF-κB是否存在调节作用及其对FKN表达的影响尚不清楚。目的:通过研究LBP、FKN在ARDS患者外周血、肺泡灌洗液(bronchoalveolar lavage fluid,BALF)中的表达变化,明确其与ARDS患者血清内毒水平、病情严重度、预后的关系,并进一步研究LBP对LPS刺激的A549细胞以及LPS诱导的ARDS大鼠动物模型中FKN的表达的影响及其细胞信号转导机制,试图阐明LBP、FKN在ARDS发病中的作用,明确参与LBP调控FKN的细胞信号转导通路,寻找出ARDS治疗的新靶点。方法:第一部分:RT-PCR检测健康对照组和ARDS患者外周血LBP、FKN m RNA,ELISA检测肺泡灌洗液、外周血LBP、FKN蛋白的表达,并研究上述指标与ARDS患者血浆内毒素水平、病情严重程度及预后的关系。第二部分:分别在使用LBP质粒DNA、LBP sh RNA质粒DNA、p38MAPK、p65NF-κB信号通路的抑制剂SB203580、SC-514进行预处理的A549细胞上,使用LPS刺激后,RT-PCR检测细胞的LBP、FKN m RNA的表达,ELISA检测细胞培养上清液中LBP、FKN蛋白,western blotting检测细胞的LBP、FKN、磷酸化p38MAPK(phospho-p38MAPK)、磷酸化p65蛋白(phospho-p65)蛋白的表达,免疫荧光染色和共聚焦显微镜(Confocal Laser Scanning Microscope,CLSM)观察phospho-p38MAPK、phospho-p65的细胞核转移的情况。免疫共沉淀(Co-Immunoprecipitation,COIP)检测LBP与phospho-p38MAPK、phospho-p65的相互作用。第三部分:在大鼠模型上,分别经尾静脉注射LBP抑制多肽LBPK95A,p38MAPK、p65NF-κB信号通路的抑制剂SB203580、SC-514对大鼠进行预处理后,再注射LPS,RT-PCR检测大鼠肺组织的LBP、FKN m RNA表达,ELISA检测大鼠肺组织匀浆和大鼠血清LBP、FKN蛋白的表达,western blotting检测大鼠肺组织匀浆中LBP、FKN、phospho-p38MAPK、phospho-p65蛋白的表达,免疫组织化学方法检测FKN蛋白在大鼠肺组织中的表达分布及变化情况。结果:第一部分:与健康组相比,ARDS患者外周血LBP m RNA,血清LBP蛋白升高,FKN m RNA和蛋白表达下降(p均小于0.05)。与轻度组相比,中度组LBP m RNA、BALF及血清LBP蛋白表达的升高,重度组进一步升高;FKN表达下降,重度组进一步下降(p均小于0.05)。死亡组患者的LBP表达较存活组升高,FKN表达较存活组下降(p均小于0.05)。血清LBP蛋白水平与内毒素定量、APACHEⅡ评分、病情严重程度成正相关(p均小于0.05,r分别为0.8878,0.8365,0.862)。血清FKN蛋白水平与内毒素定量、APACHE II评分、病情严重程度成负相关(p均小于0.05,r分别为-0.7341,-0.7726,-0.695)。LBPm RNA与FKN m RNA,肺泡灌洗液、血清中的LBP蛋白与FKN蛋白成负相关(P均小于0.05,r分别为-0.8378,-0.7908,-0.8572)。LBP作为评估ARDS患者死亡的诊断指标,其敏感性与APACHEⅡ评分一致,但特异性优于APACHEⅡ评分。第二部分:与对照组相比,LPS刺激后,细胞LBP m RNA和蛋白升高,FKN m RNA和蛋白表达下降,LBP质粒DNA使FKN m RNA和蛋白表达进一步下降,LBP sh RNA质粒DNA、SB203580、SC-514均能抑制LPS介导的FKN m RNA和蛋白表达的下降;LPS使细胞的phospho-p38MAPK、phospho-p65蛋白的表达升高,LBP质粒DNA能促进这一作用,LBP sh RNA质粒DNA、SB203580和SC-514预处理能分别抑制细胞phospho-p38MAPK、phospho-p65蛋白的升高表达(p均小于0.05,n=6);免疫荧光染色及CLSM观察显示,使用LPS后A549细胞内phospho-p38MAPK、phospho-p65蛋白由细胞浆转移至细胞核,LBP质粒DNA促进phospho-p38MAPK、phospho-p65蛋白的核转移,使用LBP sh RNA质粒DNA、SB203580和SC-514预处理能分别抑制phospho-p38MAPK、phospho-p65蛋白的核转移。COIP检测到LBP与phospho-p38MAPK、phospho-p65存在相互作用。第三部分:与对照组相比,LPS使大鼠LBP m RNA和蛋白升高,FKN m RNA和蛋白表达下降,大鼠肺组织匀浆的phospho-p38MAPK、phospho-p65蛋白表达上升,LBPK95A能抑制ARDS大鼠肺组织匀浆和血清的LBP蛋白的升高,SB203580、SC-514的预处理能抑制ARDS大鼠肺组织匀浆和血清的FKN蛋白的下降,LBPK95A、SB203580和SC-514预处理能分别抑制ARDS大鼠肺组织匀浆phospho-p38MAPK、phospho-p65蛋白的升高表达(p均小于0.05,n=10);免疫组织化学染色观察到FKN蛋白主要分布于肺泡上皮细胞,LPS使大鼠肺泡上皮细胞FKN蛋白表达减少,LBPK95A、SB203580、SC-514的预处理能抑制LPS对FKN蛋白的下调作用。结论1.ARDS患者外周血LBP m RNA、肺泡灌洗液、血清的LBP蛋白水平升高,FKN m RNA和蛋白水平下降,LBP、FKN与ARDS患者的内毒素水平、病情严重程度相关,LBP可以作为一个评估ARDS患者死亡预后的新指标。2.在ARDS细胞和动物模型中,LBP可能通过激活p38MAPK、NF-κB信号通路,下调FKN的表达,参与了LPS介导了ARDS的过程。3.使用LBP抑制多肽LBPK95A进行干预,能减轻LPS引起的肺损伤,可能将成为ARDS治疗的一个新靶点。
[Abstract]:Background: Acute respiratory distress syndrome (ARDS) is a common clinical critical disease with high mortality. The inflammatory response mediated by endotoxemia is one of the common causes of the pathogenesis of ARDS. The lipopolysaccharide (lipopolysaccharide, LPS) is the main component of the endotoxin. Binding protein, LBP) is a glycoprotein that exists in normal human and animal serum, increases in the acute stage of inflammatory response, and participates in a variety of inflammatory factors that regulate the LPS-LBP complex formed by the combination of.LPS and LBP to activate mononuclear cells, macrophages and other cells, CD14/ TLR4 receptors, Toll like receptor 4 (Toll-like receptor4, TLR4). After activating the intracellular signal transduction of LPS, the transduction pathway is carried out in two directions, namely, the mitogen activated protein kinase (MAPK) pathway and nuclear factor kappa B (nuclear factor- kappa B, NF- kappa B) pathway. KN) is the only member of the chemokine CX3C family. On the one hand, FKN plays an important role in the recruitment of inflammatory cells on the vascular wall and the injury of endothelial cells, while the other side plays an anti-inflammatory role by inhibiting the inhibition of apoptosis. Currently, LBP activates MAPK, and does NF- kappa B have a regulatory effect and its effect on the expression of FKN. Objective: To study the changes in expression of LBP, FKN in peripheral blood of ARDS and bronchoalveolar lavage fluid (BALF) in patients with ARDS, and to clarify the relationship with the level of endotoxin, severity and prognosis of serum ARDS patients, and further study the tables of LBP on LPS stimulated A549 cells and LPS induced rat models. The effect of Da and its cell signal transduction mechanism, trying to elucidate the role of LBP and FKN in the pathogenesis of ARDS, clearly participating in the cellular signal transduction pathway of FKN by LBP, and finding new targets for the treatment of ARDS. Method: Part 1: RT-PCR detection of LBP in the peripheral blood of the healthy control and ARDS patients, FKN m RNA, the alveolar lavage fluid, the peripheral blood, and the peripheral blood, The expression of protein, and the relationship between the plasma endotoxin level, severity and prognosis of ARDS patients. The second part: the LBP plasmid DNA, the LBP sh RNA plasmid DNA, p38MAPK, the p65NF- kappa B signaling pathway inhibitor SB203580, the SC-514 proceed to detect the cells The expression of BP, FKN m RNA, ELISA was used to detect LBP, FKN protein, LBP, FKN, phosphorylated, phosphorylated protein, and immunofluorescence staining and confocal microscopy. The nuclear transfer of o-p65. Co-Immunoprecipitation (COIP) was used to detect the interaction between LBP and phospho-p38MAPK and phospho-p65. The third part: in the rat model, the LBP inhibition of polypeptide LBPK95A, p38MAPK, p65NF- kappa B signal through the rat model, the inhibitor SB203580, after the rat was pretreated, and then the rats were pretreated, and then the rats were pretreated, and then the rats were pretreated and then the rats were pretreated. The expression of LBP, FKN m RNA in lung tissue of rats was detected by injection of LPS and RT-PCR. ELISA was used to detect the expression of LBP, FKN protein in rat lung homogenate and rat serum. Western blotting was used to detect the expression of LBP, expression and protein in lung homogenate of rats. Results: the first part: compared with the health group, the peripheral blood LBP m RNA, the serum LBP protein and the FKN m RNA and protein expression decreased (p less than 0.05). Compared with the mild group, the moderate group LBP m RNA, the increase of the expression of serum albumin, the increase of the weight group, the decrease of the expression and the further decrease of the severe group. The expression of LBP in the death group was higher than that in the survival group, and the expression of FKN was lower than that of the survival group (P was less than 0.05). The serum LBP protein level was positively correlated with the endotoxin, APACHE II score, and the severity of the disease (P was less than 0.05, R was 0.8878,0.8365,0.862). Serum FKN protein level and endotoxin quantitative, APACHE II score, disease, disease, and disease. The degree of severity was negatively correlated (P was less than 0.05, R was -0.7341, -0.7726, -0.695).LBPm RNA and FKN m RNA, alveolar lavage fluid, and LBP protein in serum was negatively correlated with FKN protein (less than 0.05, respectively. The specificity was better than the APACHE II score. Second: compared with the control group, the cell LBP m RNA and protein increased, the FKN m RNA and protein expression decreased after LPS stimulation. LBP plasmid DNA made FKN m and protein expression further decreased. The expression of phospho-p38MAPK, phospho-p65 protein increased, and LBP plasmid DNA could promote this effect. LBP sh RNA plasmid DNA, SB203580 and SC-514 pretreatment could inhibit cell phospho-p38MAPK, phospho-p65 protein increased expression (all less than 0.05). K, phospho-p65 protein is transferred from cytoplasm to nucleus, and LBP plasmid DNA promotes nuclear transfer of phospho-p38MAPK, phospho-p65 protein, LBP sh RNA plasmid DNA, SB203580 and SC-514 pretreatment can inhibit respectively. Third Part: compared with the control group, LPS increased LBP m RNA and protein, FKN m RNA and protein expression decreased, the expression of phospho-p38MAPK, phospho-p65 protein in lung homogenate of rats increased, LBPK95A could inhibit the increase of LBP protein in lung homogenate and serum of ARDS rats. The decrease of FKN protein in serum and serum, LBPK95A, SB203580 and SC-514 pretreatment could inhibit the expression of phospho-p38MAPK and phospho-p65 protein in the lung homogenate of ARDS rats (p less than 0.05, n=10). Immunohistochemical staining showed that FKN protein was mainly distributed in the alveolar skin cells, and the FKN protein expression in alveolar epithelial cells of rats was reduced. LBPK95A, SB203580, SC-514 preconditioning can inhibit the downregulation of LPS to FKN protein. Conclusion 1.ARDS patients' peripheral blood LBP m RNA, alveolar lavage fluid, the level of LBP protein in serum, FKN m and protein levels decline, and are related to the level of endotoxin and the severity of the disease. In the ARDS cell and animal model, the new index of death prognosis, LBP may reduce the expression of FKN by activating p38MAPK, NF- kappa B signaling pathway, and participates in LPS mediated ARDS process.3. using LBP inhibition polypeptide LBPK95A, which can reduce lung injury caused by LPS, and may become a new target for the treatment of ARDS.
【学位授予单位】：暨南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R563.8

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