本文選題：急性呼吸窘迫綜合征 + 脂多糖結(jié)合蛋白��； 參考：《暨南大學(xué)》2017年博士論文

【摘要】：研究背景:急性呼吸窘迫綜合征(Acute respiratory distress syndrome,ARDS)是臨床上常見的危重癥,病死率高,內(nèi)毒素血癥介導(dǎo)的炎性反應(yīng)是ARDS發(fā)病的常見原因之一。脂多糖(lipopolysaccharide,LPS)是內(nèi)毒素的主要成份。脂多糖結(jié)合蛋白(lipopolysaccharide binding protein,LBP)是存在于正常人和動物血清中的一種糖蛋白,在炎癥反應(yīng)的急性期升高,參與多種炎癥因子的調(diào)控。LPS與LBP結(jié)合形成的LPS-LBP復(fù)合物激活單核細(xì)胞、巨噬細(xì)胞和其它細(xì)胞上的CD14/TLR4受體,Toll樣受體4(Toll-like receptor4,TLR4)激活后可介導(dǎo)LPS胞內(nèi)信號轉(zhuǎn)導(dǎo),轉(zhuǎn)導(dǎo)通路向兩個(gè)方向進(jìn)行,即絲裂原活化蛋白激酶(mitogen activated protein kinase,MAPK)通路和核因子-κB(nuclear factor-κB,NF-κB)通路。MAPK、NF-κB信號通路活化后通過調(diào)節(jié)下游炎癥因子的表達(dá)誘發(fā)炎癥反應(yīng)。Fractalkine(FKN)是趨化因子CX3C家族的唯一成員,一方面FKN于炎性細(xì)胞在血管壁上的募集和內(nèi)皮細(xì)胞的損傷具有重要的作用,而另一方其通過對細(xì)胞凋亡的抑制作用起到抗炎作用。目前LBP對LPS激活MAPK、NF-κB是否存在調(diào)節(jié)作用及其對FKN表達(dá)的影響尚不清楚。目的:通過研究LBP、FKN在ARDS患者外周血、肺泡灌洗液(bronchoalveolar lavage fluid,BALF)中的表達(dá)變化,明確其與ARDS患者血清內(nèi)毒水平、病情嚴(yán)重度、預(yù)后的關(guān)系,并進(jìn)一步研究LBP對LPS刺激的A549細(xì)胞以及LPS誘導(dǎo)的ARDS大鼠動物模型中FKN的表達(dá)的影響及其細(xì)胞信號轉(zhuǎn)導(dǎo)機(jī)制,試圖闡明LBP、FKN在ARDS發(fā)病中的作用,明確參與LBP調(diào)控FKN的細(xì)胞信號轉(zhuǎn)導(dǎo)通路,尋找出ARDS治療的新靶點(diǎn)。方法:第一部分:RT-PCR檢測健康對照組和ARDS患者外周血LBP、FKN m RNA,ELISA檢測肺泡灌洗液、外周血LBP、FKN蛋白的表達(dá),并研究上述指標(biāo)與ARDS患者血漿內(nèi)毒素水平、病情嚴(yán)重程度及預(yù)后的關(guān)系。第二部分:分別在使用LBP質(zhì)粒DNA、LBP sh RNA質(zhì)粒DNA、p38MAPK、p65NF-κB信號通路的抑制劑SB203580、SC-514進(jìn)行預(yù)處理的A549細(xì)胞上,使用LPS刺激后,RT-PCR檢測細(xì)胞的LBP、FKN m RNA的表達(dá),ELISA檢測細(xì)胞培養(yǎng)上清液中LBP、FKN蛋白,western blotting檢測細(xì)胞的LBP、FKN、磷酸化p38MAPK(phospho-p38MAPK)、磷酸化p65蛋白(phospho-p65)蛋白的表達(dá),免疫熒光染色和共聚焦顯微鏡(Confocal Laser Scanning Microscope,CLSM)觀察phospho-p38MAPK、phospho-p65的細(xì)胞核轉(zhuǎn)移的情況。免疫共沉淀(Co-Immunoprecipitation,COIP)檢測LBP與phospho-p38MAPK、phospho-p65的相互作用。第三部分:在大鼠模型上,分別經(jīng)尾靜脈注射LBP抑制多肽LBPK95A,p38MAPK、p65NF-κB信號通路的抑制劑SB203580、SC-514對大鼠進(jìn)行預(yù)處理后,再注射LPS,RT-PCR檢測大鼠肺組織的LBP、FKN m RNA表達(dá),ELISA檢測大鼠肺組織勻漿和大鼠血清LBP、FKN蛋白的表達(dá),western blotting檢測大鼠肺組織勻漿中LBP、FKN、phospho-p38MAPK、phospho-p65蛋白的表達(dá),免疫組織化學(xué)方法檢測FKN蛋白在大鼠肺組織中的表達(dá)分布及變化情況。結(jié)果:第一部分:與健康組相比,ARDS患者外周血LBP m RNA,血清LBP蛋白升高,FKN m RNA和蛋白表達(dá)下降(p均小于0.05)。與輕度組相比,中度組LBP m RNA、BALF及血清LBP蛋白表達(dá)的升高,重度組進(jìn)一步升高;FKN表達(dá)下降,重度組進(jìn)一步下降(p均小于0.05)。死亡組患者的LBP表達(dá)較存活組升高,FKN表達(dá)較存活組下降(p均小于0.05)。血清LBP蛋白水平與內(nèi)毒素定量、APACHEⅡ評分、病情嚴(yán)重程度成正相關(guān)(p均小于0.05,r分別為0.8878,0.8365,0.862)。血清FKN蛋白水平與內(nèi)毒素定量、APACHE II評分、病情嚴(yán)重程度成負(fù)相關(guān)(p均小于0.05,r分別為-0.7341,-0.7726,-0.695)。LBPm RNA與FKN m RNA,肺泡灌洗液、血清中的LBP蛋白與FKN蛋白成負(fù)相關(guān)(P均小于0.05,r分別為-0.8378,-0.7908,-0.8572)。LBP作為評估ARDS患者死亡的診斷指標(biāo),其敏感性與APACHEⅡ評分一致,但特異性優(yōu)于APACHEⅡ評分。第二部分:與對照組相比,LPS刺激后,細(xì)胞LBP m RNA和蛋白升高,FKN m RNA和蛋白表達(dá)下降,LBP質(zhì)粒DNA使FKN m RNA和蛋白表達(dá)進(jìn)一步下降,LBP sh RNA質(zhì)粒DNA、SB203580、SC-514均能抑制LPS介導(dǎo)的FKN m RNA和蛋白表達(dá)的下降;LPS使細(xì)胞的phospho-p38MAPK、phospho-p65蛋白的表達(dá)升高,LBP質(zhì)粒DNA能促進(jìn)這一作用,LBP sh RNA質(zhì)粒DNA、SB203580和SC-514預(yù)處理能分別抑制細(xì)胞phospho-p38MAPK、phospho-p65蛋白的升高表達(dá)(p均小于0.05,n=6);免疫熒光染色及CLSM觀察顯示,使用LPS后A549細(xì)胞內(nèi)phospho-p38MAPK、phospho-p65蛋白由細(xì)胞漿轉(zhuǎn)移至細(xì)胞核,LBP質(zhì)粒DNA促進(jìn)phospho-p38MAPK、phospho-p65蛋白的核轉(zhuǎn)移,使用LBP sh RNA質(zhì)粒DNA、SB203580和SC-514預(yù)處理能分別抑制phospho-p38MAPK、phospho-p65蛋白的核轉(zhuǎn)移。COIP檢測到LBP與phospho-p38MAPK、phospho-p65存在相互作用。第三部分:與對照組相比,LPS使大鼠LBP m RNA和蛋白升高,FKN m RNA和蛋白表達(dá)下降,大鼠肺組織勻漿的phospho-p38MAPK、phospho-p65蛋白表達(dá)上升,LBPK95A能抑制ARDS大鼠肺組織勻漿和血清的LBP蛋白的升高,SB203580、SC-514的預(yù)處理能抑制ARDS大鼠肺組織勻漿和血清的FKN蛋白的下降,LBPK95A、SB203580和SC-514預(yù)處理能分別抑制ARDS大鼠肺組織勻漿phospho-p38MAPK、phospho-p65蛋白的升高表達(dá)(p均小于0.05,n=10);免疫組織化學(xué)染色觀察到FKN蛋白主要分布于肺泡上皮細(xì)胞,LPS使大鼠肺泡上皮細(xì)胞FKN蛋白表達(dá)減少,LBPK95A、SB203580、SC-514的預(yù)處理能抑制LPS對FKN蛋白的下調(diào)作用。結(jié)論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與ARDS患者的內(nèi)毒素水平、病情嚴(yán)重程度相關(guān),LBP可以作為一個(gè)評估ARDS患者死亡預(yù)后的新指標(biāo)。2.在ARDS細(xì)胞和動物模型中,LBP可能通過激活p38MAPK、NF-κB信號通路,下調(diào)FKN的表達(dá),參與了LPS介導(dǎo)了ARDS的過程。3.使用LBP抑制多肽LBPK95A進(jìn)行干預(yù),能減輕LPS引起的肺損傷,可能將成為ARDS治療的一個(gè)新靶點(diǎn)。
[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.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R563.8

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