【作者】 刘敏；

【导师】 王怀立；

【作者基本信息】 郑州大学 ， 儿科学， 2013， 硕士

【摘要】 背景和目的肺炎链球菌溶血素(pneumolysin, PLY)是由肺炎链球菌(Streptococcus pneumoniae, S.pn)溶解后释放的主要毒力因子之一。PLY具有着多重的功能,可以破坏血脑屏障的细胞间紧密连接、引起细胞的自我溶解、刺激炎症因子的释放及诱导细胞凋亡等,导致脑组织的严重损伤,与患儿的预后密切相关,在肺炎链球菌脑膜炎发生发展中起关键作用。因此进一步深入研究PLY导致脑损伤的机制,并采用相应措施阻断其导致脑损伤的过程具有重要的意义。髓样细胞触发受体1(Triggering receptor expressed on myeloid cells-1TREM-1)是最近发现的表达于中性粒细胞和单核／巨噬细胞表面的受体,作为感染性疾病中炎症激发和级联放大的关键物质,它主要通过受体识别模式(TLRs)刺激炎症因子的释放(包括TNF-α、IL-6、IL-8等),进而引起组织损伤,虽然其自然配体尚未发现,但在细菌和真菌感染中可发现其表达增高。目前已有研究证实TREM-1在脓毒症、肺炎、急性化脓性胆管炎等急性细菌感染性疾病中中性粒细胞、单核及巨噬细胞表面表达上调,但在神经系统感染性疾病尤其是婴幼儿细菌性脑膜炎中的研究报道不多。为此本实验通过颈内动脉注射PLY制作感染性脑损伤大鼠模型,并检测TREM-1在PLY致脑损伤模型中不同时间点的表达水平,并与脑损伤的指标进行相关性分析,探讨其在PLY在致脑损伤过程中的初步作用机制及意义。材料与方法将64只幼鼠随机分为2组：肺炎球菌溶血素组(即PLY组,n=32),经左侧颈内动脉注射0.1mL(8μg)PLY;生理盐水对照组(即NS组,n=32),经左侧颈内动脉注射等体积的生理盐水。两组按注射后不同时间点分为4h、6h、12h、24h4个亚组,平均每个亚组8只幼鼠。于预定时间点处死大鼠,制备脑组织匀浆液及石蜡切片标本。免疫组织化学技术(SP法)测定脑内皮层、海马组织NSE、 GFAP蛋白表达情况,酶联免疫吸附法(ELISA)检测脑组织TREM-1、Clq、TNF-αIL-6蛋白表达情况。所有数据采用SPSS17.0软件进行统计分析,多组样本均数比较采用单因素方差分析,两样本间相关性比较采用Pearson相关分析,显著性水准为α<0.05。结果1.形态学及行为学改变：肉眼观PLY组大鼠左侧眼球凹陷、坏死,眼睑完全闭合,眼角处可见局部出血,体重减轻可达10%-15%。解剖学观察左侧脑体积较右侧增大,脑组织肿胀,脑膜紧张,脑血管充血明显。HE染色光镜下观察PLY组新生血管形成,脑血管间隙增宽,神经元细胞、胶质细胞不同程度肿胀、空泡变性,NS组大鼠没有上述变化。2.PLY组TREM-1水平在4h达顶峰,6h仍较高,具有统计学差异(p<0.05),12h与24hTREM-1水平迅速下降,与NS对照组相比无统计学差异。C1q水平在6h达顶峰,12h仍较高,具有统计学意义(p<0.05),4h与24hClq与NS对照组相比无统计学差异。TNF-α及IL-6水平在各时间点均明显高于对照组,并在24h组达到顶峰,差异具有统计学意义(p<0.05)。TREM-1水平与Clq表达水平不具有相关性,相关系数r=0.052,P=0.778; TREM-1水平与TNF-α表达水平具有相关性。相关系数r=0.737,P<0.05;TREM-1水平与IL-6表达水平具有相关性,相关系数r=0.659,P<0.05。3. TREM-1水平与GFAP表达水平具有相关性,相关系数r=0.570,P<0.05；TREM-1表达水平与NSE水平具有相关性,相关系数r=0.641,P<0.05。结论1.PLY可以引起TREM-1的表达,并可由此参与炎症反应过程,促进炎症因子释放。2.TREM-1参与PLY所引起脑组织损伤的病理发展过程。3.在PLY引起脑损伤的过程中TREM-1并不通过经典补体途径产生作用。更多还原

【Abstract】 Background and objectivePneumolysin (PLY) is released after Streptococcus pneumoniae (S.pn) dissolved. as a major virulence factor of S.pn, PLY has multiple functions, it can disrupt the tight junction between cells which constitute the blood-brain barrier, causing cells self-dissolution, stimulate the release of inflammatory factors, and inducing cell apoptosis, then resulted in serous brain damage.,which is closely related to the prognosis of the children patient have pneumococcal meningitis, and Play a key role in the development of pneumococcal meningitis. Therefor,further study on the mechanisms of how PLY cause the brain damage and adopt appropriate measures to block the process of PLY caused brain damage is of a great significance.TREM-1is a recently discovered receptor which expressed on neutrophil and monocyte/macrophage.As a key substance stimulate and amplify the inflammation cascade after an infection disease, TREM-1is mainly through the receptor recognition model (TLRs) to stimulate the release of the pro-inflammatory cytokines (including the TNF-a, IL-6, IL-8etc.), causing tissue damage. Although its natural ligand has not yet been found, but in the bacterial and fungal infection disease can be found in the higher. To date, the study confirmed that in sepsis, pneumonia, acute suppurative cholangitis and other acute bacterial infectious diseases,TREM-1was found expressed on surface of neutrophils, monocytes and macrophages, but report in the central nervous system infectious disease especially in infants and young children with bacterial meningitis is not many.In this experiment,we produced an infectious brain injury rat model through injecting the PLY into the rat’s left internal carotid artery, then detect the TREM-1expression levels at different time points, and the correlation with the brain injury.To explore the mechanism of PLY played in the inflammatory reaction in vivo,and find how does the PLY play a role during the inflammatory process of brain injury.Materials and methodsWe randomly divided64infant rats into two groups,the pneumolysin group(PLY group, n=32):In the left internal carotid artery with a injection of0.1mL (8μg) PLY; control group (group NS, n=32):inject the left internal carotid artery of rat with an equal volume of saline. Both two groups are divided into4h,6h,12h,24h4subgroups according to the different time points after injection into PLY, each subgroup include8infant rats. At each time point we execute the8infant rats, after the execution of each rat,the sample of brain tissue were prepared by tissue homogenate and make the paraffin sections. The expression of the NSE protein and GFAP protein in the cerebral cortex and hippocampus were measured by the immunohistochemistry (SP), And enzyme linked immunosorbent assay (ELISA) were used to detect the expression levels of TREM-1, C1q, TNF-α, IL-6protein in brain tissue. All data were analyzed by SPSS17.0software. Several groups of samples were compared with single factor analysis of variance, correlation between the two samples was analyzed by Pearson correlation analysis,the significance level is a<0.05.Result1. Morphological and behavioral change:In the PLY group the left eye of the infant rat we can see the enophthalmos, necrosis,the eyelid completely closed,and even in the corner of eye we can see a visible bleeding.The weight loss of the infant rat was up to10%-15%. In the anatomical observation,we can see the left brain volume is bigger than the right brain, with the brain swelling, dural tension, cerebral vascular congestion significantly. In PLY group, After the HE staining under light microscope,the formation of new blood vessels,and the cerebral vascular gap widened, neurons, different degrees of glial cell swelling,and vacuolar degeneration can be seen. While in NS group none of the above changes could be seen.2. The level of TREM-1in the PLY group reached a peak in the4h time point, in the6h time point,the level of TREM-1is still high, both groups have the statistical difference with the control group(P<0.05), while decreased levels of TREM-1were found in12h and24h subgroups, and when compared with the control group there is no significant difference.The Clq levels reached the peak at6h time point, in12h time point the level is still high, and have a statistical significance when compared with the control group(P<0.05).While in4h and24h time point,the levels of Clq have no significant difference between the PLY and the control group. At each time point levels of TNF-a and IL-6were all significantly higher than those in the control group, and reached a peak in the24h time point, the difference was statistically significant (P<0.05).The level of TREM-1and Clq were uncorrelated, the correlation coefficient is r=0.052,(P=0.778);the TNF-α level is correlated with TREM-l,and the correlation coefficient is r=0.737,(P<0.05);the level of IL-6is correlated with TREM-1,the correlation is r=0.659,(P<0.05).3. The level of GFAP and TREM-1were correlated, the correlation coefficient is r=0.570,(P<0.05); correlation between NSE level and TREM-llevel was found, the correlation coefficient between them is r=0.641,(P<0.05).Conclusion1. PLY can up-regulate the expression of TREM-1, and thus participate in the inflammatory process, to promote the release of inflammatory cytokines.2. TREM-1is involved in the pathological process of brain damage induced by PLY.3. The effect of TREM-1in the brain damage caused by PLY is not through the classical complement pathway.更多还原