【作者】 史小娟；

【导师】 罗自强；

【作者基本信息】 中南大学 ， 生理学， 2008， 硕士

【摘要】 第一章MK801对小鼠油酸型肺损伤的保护作用研究背景急性呼吸窘迫综合征(acute respiratory distress syndrome,ARDS)是临床常见危重疾病,发病机制尚未完全阐明。肺内中性粒细胞、巨噬细胞的滞留和激活在ARDS的发生发展中起重要作用。中性粒细胞、巨噬细胞可释放谷氨酸。Said等人发现谷氨酸离子型受体特异性激动剂N-甲基-D-门冬氨酸(N-methyl-D-aspartate,NMDA)可引起离体灌流肺发生高通透性肺水肿。近年来本室的研究还发现NMDA受体阻断剂MK-801对脂多糖(Lipopolysaccharide,LPS)和高氧等引起的肺损伤具有明显的保护作用,这些研究进一步提示内源性谷氨酸(Glutamate,Glu)的大量释放过度激活NMDA受体参与急性肺损伤的发生发展过程。但为证明内源性Glu的大量释放过度激活NMDA受体在急性肺损伤的发生中具有普遍意义,尚有待在不同模型上进一步证实。方法(1)建立小鼠急性油酸肺损伤模型;(2)测量肺湿重和干重的比值(W/D)并观察肺组织病理变化,以反映肺组织的损伤程度;(3)测定支气管肺泡灌洗液中蛋白和白细胞含量,以反映支气管肺泡毛细血管通透性变化;(4)生化测定肺组织髓过氧化物酶(MPO),反映氧化损伤时的肺炎性改变;(5)RT-PCR和免疫组化分别检测CTP:磷酸胆碱二胞苷酰基转移酶α(CTP:phosphocholinecytidylyltransferase alpha,CCTα)mRNA和蛋白水平,反映CCTα转录合成的变化。结果油酸(0.1 ml/kg)尾静脉注射4 h可引起肺组织肺指数、肺湿干重比W/D增加、肺组织MPO活性增加,支气管肺泡灌洗液中蛋白和白细胞含量明显增加,并引起肺组织CCTαmRNA和蛋白水平的明显减低,病理切片示水肿、炎症渗出明显。NMDA受体的阻断剂MK-801(0.1mg/kg,ip)预处理30 min可减轻油酸引起的上述变化。结论油酸引起明显以炎性渗出为主的急性肺损伤,肺内NMDA受体的激活参与油酸急性肺损伤的发生,并引起CCTα表达的下调。第二章NMDA抑制CCTα表达的转录调控机制研究背景CTP:磷酸胆碱二胞苷酰基转移酶(CTP:phosphocholinecytidylyltransferase alpha,CCTα)是磷脂酰胆碱(phosphatidylcholine,PC)合成中的关键调控酶。CCTα基因近端启动子区包含多个与细胞生长、分化密切相关的转录因子Sp1(specificity protein 1)结合位点。探讨Sp1在CCTα启动转录中的作用具有一定的意义。在急性肺损伤(acute lung iniury,ALI)及ARDS等肺部损伤疾病中,肺表面活性物质(pulmonary surfactant,PS)系统功能的异常并参与肺损伤的发生过程。本室前期研究证明一定剂量NMDA作用后,肺Ⅱ型上皮细胞合成CCT mRNA水平、CCTα蛋白表达水平均下降。本研究旨在观察NMDA受体激活后Sp1的改变,探讨NMDA抑制CCTα表达的分子机制。方法(1)萤光素酶报告基因活性检测CCTα启动子活性;(2)荧光定量PCR定量检测Sp1表达质粒后CCTαmRNA;(3)Western blot检测A549细胞转染Sp1表达质粒后CCTα蛋白含量表达水平;(4)Western blot检测A549细胞经NMDA处理后Sp1蛋白含量表达水平;(5)荧光定量PCR定量检测A549细胞经NMDA处理后Sp1mRNA水平;(6)EMSA检测CCTα启动子上Sp1的DNA结合活性及NMDA处理对其影响。结果转染Sp1真核表达质粒30 h后,A549细胞CCTα核心启动子报告基因相对萤光素酶活性增加,A549细胞CCTαm RNA表达水平表达水平增高。转染Sp1真核表达质粒36 h后,CCTα蛋白表达水平表达水平增高。A549细胞经NMDA处理8 h后,Sp1蛋白表达明显下降,并呈时间、剂量依赖。Sp1 mRNA水平没有明显的改变。EMSA检测显示,NMDA处理后A549细胞的核蛋白中转录因子Sp1与DNA的结合活性下调。结论转录因子Sp1促进A549细胞CCTα的启动子活性,增加CCTα的合成表达。NMDA通过下调核内Sp1蛋白与DNA结合活性而抑制CCTα的表达。更多还原

【Abstract】 Chapter 1 Effect of MK-801 on oleic acid-induced acute lung injury in miceBackground Acute respiratory distress syndrome(ARDS)is a common clinical syndrome.The exact pathogenesis of ARDS,however, is not well characterized.Pulmonary neutrophil sequestration and the activation of neutrophils and alveolar macrophages play key roles in the pathogenesis of ARDS.It has been shown that glutamate released from activated neutrophils acts as a source of excitotoxic injury of other cells. Both macrophages and activated neutrophils release glutamate.Said et al.reported that administration of high concentrations of glutamate or glutamate agonist NMDA can elicit an acute,high-permeability pulmonary edema.In recent years,we find that the NMDA receptor antagonist MK-801(dizocilpine)attenuates lipopolysaccharide(LPS) and hyperoxia-induced lung injury.These findings suggested that lung injury(caused by LPS and hyperoxia)could be mediated partly by endogenous glutamate release and toxic overactivation of NMDA receptors.Therefore,glutamate receptors may serve novel,yet to-be characterized injury factor during acute lung injury.Further studies in different animal models of lung injury are required before assuming their universal significance.Methods(1)Oleic acid(0.1ml/kg)was injected via the caudal vein to induce acute lung injury model in mice.(2)Lung wet-to-dry weight ratio (W/D)and histopathological examination were used to evaluate the severity of pulmonary edema.(3)Lung tissue myeloperoxidase(MPO) activity was quantified by using commercial kits as a reflection of neutrophil sequestration.(4)Total neutrophil count and total protein concentration in bronchoalveolar lavage fluid(BALF)were determined to reflect changes in alveolar-capillary permeability.(5)The mRNA level of CCTαin the lung was measured by RT-PCR,and the expression of CCTαprotein was semi-quantified by immunohistochemistry. Results(1)Oleic acid-induced increases in the concentration of total protein and the numbers of neutrophils in BALF were significantly attenuated in MK-801(0.1mg/kg,ip)pretreated mice;(2)MK-801 pretreatment also resulted in a significant protection in lung tissue tested against oleic acid-induced acute lung injury via decreasing W/D ratio, MPO activity.(3)The CCTαexpression decreased in oleic acid-induced lung injury model,both at protein and mRNA levels. MK-801 pretreatment attenuated oleic acid-induced down-regulation of CCTαexpression in the lung.Conclusion Oleic acid caused an acute lung damage characterized by severe edema formation and pulmonary inflammation in mice. Endogenous NMDA receptor activation may play an important role in mediating oleic acid-induced lung injury.In this model,expression of CCTαwas decreased. Chapter 2 Study on the transcriptional regulation mechanism of CTP:phosphocholine cytidylyltransferase alpha expression inhibition by NMDABackground CTP:phosphocholinecytidylyltransferase alpha(CCTα) is the major rate-limiting enzyme for phosphatidylcholine(PC)synthesis. The promoter of CCTαis GC rich,and contains several binding sites for the specificity protein 1(Sp1)transcription factor,which is thought to play an important role in regulating cell growth and differentiation.The dysfunction of pulmonary surfactant(PS)is thought to be a important factor during the development of acute lung injury(including ARDS). Previous work in our group has demonstrated NMDA can repress CCTαmRNA and protein expression in pulmonary alveolar typeⅡcells.In this study,we examined the changes in Sp1 expression after NMDA treatment and the molecular mechanism of transcriptional regulation of inhibition of CCTαexpression by NMDA.Methods(1)CCTαpromotor activity was measured using Dual-luciferase reporter assay system.(2)CCTαmRNA expression was measured by RT-PCR after 30 h of transfection with Sp1(pEVR2-Sp1) expression plasmid.(3)CCTαprotein level was measured by Western blot after 36 h of transfection with pEVR2-Sp1 plasmid.(4)Sp1 protein in nuclear extracts after NMDA treatment was measured by Western blot.Sp1 mRNA was measured by RT-PCR,and Sp1 DNA binding activity was measured by EMSA.Results Transfection of the A549 cells with the Sp1 expression plasmid,pEVR2-Sp1,resulted in an increase in promoter activity,and as well as increases in CCTαmRNA and protein.Treatment of A549 cells with NMDA for 8h,the level of Sp1 protein decreased in a dose- and time-dependent manner in nuclear protein.But the level of Sp1 mRNA did not change significantly.EMSA showed that NMDA inhibited the DNA binding activity of Splin A549 cells.Conclusion Sp1 promotes the CCTαpromoter acticity and increase expression of CCTαin A549 cells.NMDA receptor activation inhibites CCTαexpression by decreaseing Sp1 protein and DNA binding activity in neuclear of A549 cells.更多还原