【作者】 李宇宁；

【导师】 郑江；

【作者基本信息】 第三军医大学 ， 临床检验诊断学， 2009， 硕士

【摘要】 脓毒症是由感染因素介导的全身炎症反应综合征(systemic inflammatory response syndrome,SIRS)。构成革兰阴性菌外膜的主要成分内毒素(lipopolysaccharide,LPS)是介导脓毒症的主要病原分子,在脓毒症的病理生理过程中发挥着重要的作用。LPS进入机体后,与单核-吞噬细胞系统细胞膜上的模式识别受体TLR4结合,将信号转入胞内,介导炎症细胞活化,释放炎症介质(如TNF-α、IL-1和IL-6等)而引发机体的炎症反应,此过程LPS不进入胞内即可启动信号转导。除此之外,LPS还可通过TLR4介导的网格蛋白依赖方式进行内化(Internalization)而进入胞内,以往一直认为这种方式与LPS的降解与代谢相关,但最近有研究表明,LPS内化与细胞活化有着密切关联,本课题组前期研究也表明,内体酸化抑制剂氯喹(Chloroquine,CQ)对LPS攻击小鼠具有明显的保护作用,其作用机制不明确。因此,探讨LPS内化过程与炎性细胞活化的关系,对进一步深入了解LPS介导脓毒症发生的病理生理机制具有重要意义。目的用内体成熟抑制剂CQ抑制RAW264.7细胞内体成熟,观察内体成熟障碍对LPS在内体的分布情况以及对LPS刺激的RAW264.7细胞活化的影响。方法(1)使用异硫氰酸荧光素(Fluorescein isothiocynate, FITC)标记LPS,激光共聚焦显微镜下观测其示踪效果。(2)以FITC-Dextran为内体pH荧光探针,在激光共聚焦显微镜下通过荧光强度变化测定内体pH,并进一步使用不同浓度的内体成熟抑制剂CQ处理细胞,换算出CQ浓度与内体pH值的对应关系。设置系列CQ浓度和尼日利亚菌素-pH梯度溶液处理LPS刺激的RAW264.7细胞,以观察内体pH变化对RAW264.7细胞中LPS的分布和细胞活化的影响。(3)使用激光共聚焦显微镜观察FITC-LPS在胞膜及胞内的分布情况。(4)用CQ预处理RAW264.7细胞后加入刺激因素LPS,分别在4h和16h收集细胞上清液,采用ELISA方法检测细胞因子TNF-a和IL-6,观察LPS刺激的RAW264.7细胞在内体成熟障碍下细胞的活化情况。结果(1)使用FITC标记LPS,经超滤方法纯化后,标记效率为33.13 mol FITC / mol LPS,激光扫描共聚焦显微镜下观察示踪效果好于商业化试剂,可以用于本课题LPS示踪。(2)使用激光共聚焦显微镜检测FITC-Dxtran荧光值,根据CQ浓度与pH的对应关系,观察到随着CQ浓度增加内体pH升高,并呈现明显的线性关系,其中,CQ在浓度为20ug/ml时将内体pH作用到pH5.07。(3) CQ的细胞毒性浓度为30ug/ml,此范围内,梯度浓度的CQ预处理RAW264.7细胞细胞之后,LPS在胞内的聚集随着CQ的浓度增加而增加,该结果与利用尼日利亚菌素-梯度pH缓冲液调节内体pH后,LPS在胞内的聚集情况结果一致。(4)经CQ预处理的RAW264.7细胞,在LPS刺激作用下, TNF-α和IL-6的释放与CQ浓度成负相关,表明内体成熟障碍对LPS刺激的细胞活化具有抑制作用。结论(1)用FITC成功标记LPS,解决了LPS示踪无商品试剂供给的问题。(2) RAW264.7细胞内体酸化成熟障碍,可导致LPS在细胞内的聚集。(3) RAW264.7细胞内体成熟障碍对LPS刺激的细胞活化具有抑制作用。更多还原

【Abstract】 IntroductionSepsis, which is systemic inflammatory response syndrome (SIRS) induced by infection, acts as a common complication in patients with extensive deep infection, severe traumas and major operations. LPS, one major component of the outer membrane of gram negative bacteria, is the most important pathogen associated molecule pattern (PAMPs) for sepsis. LPS binds to Toll like receptor 4(TLR4) on the membrane of monocytes and macrophages to induce pro inflammatory signal transduction and the release of cytokines such as TNF- a and IL-6, which ultimately results in the occurrence of sepsis. It is generally considered that LPS does not enter the cell to initiate the signaling cascade. Internalization of LPS functions as a process of degradation. However, the effects of LPS internalization on LPS inducing cell-activation has been re-realised recently, the investigation of which may enrich our understandings on the relationship between LPS and host responses and provide a new therapeutic target for the treatment of sepsis.ObjectTo observe the distribution of LPS and to analyze effects of endosome maturation arrest on LPS inducing activation of RAW264.7 cells after the block of endosomes maturation in RAW264.7 cells with an inhibitors called chloroquine (CQ).Methods(1) Fluorescein isothiocynate was chose to label LPS. The labeling efficiency was measured and the tracy effectiveness of labeled LPS was observed under a confocal laser scaning microscopy;(2) A standard curve of pH-fluorescence intensity was made by using FITC-Dextran as the probe and Nigericin to fix the pH value in endosomes. Different concentration of CQ was added to preterit cells and its effects on the maturation of endorse was detected by measuring the fluorescence intensity of FITC-Dextrin;(3) The same concentration of CQ was used to treat the RAW264.7 cell, the distribution of LPS in cell membrane and the inner of the cell. was observed.(4) The same concentration of CQ was used to treat the RAW264.7 cell cell activation status was analyzed by checking the IL-6 and TNF-a levels with ELISA methods.Results(1) The labling and purification method is efficient with a labeling rate at 33.13 mol FITC / mol LPS. The labled LPS was well traced under the confocal laser scaning microscopy; (2) The standard curve of pH-fluorescence intensity is stable and reliable, CQ could inhibited endosome maturation/acidification in a dose dependent manner; (3) CQ increased the accumulation of LPS inside RAW 264.7 cells in a dose dependent manner; (4) CQ inhibited the releasing of cytokines such as TNF-a and IL-6 in RAW264.7 cells in a dose dependent manner.Conclusions(1) LPS was successfully labeled with FITC, the efficiency of which is better than purchased ones.(2) With the block of endosome acidification/maturation in the RAW264.7 cell by CQ, more LPS could accumulated in cells.(3) The block of endosome acidification in RAW264.7 cell can lead to the inhibition of cell activation.更多还原