【作者】 李金凤；

【导师】 罗自强；

【作者基本信息】 中南大学 ， 生理学， 2008， 硕士

【摘要】 第一章人外周血中性粒细胞不同分离方法的比较目的:从血液中分离中性粒细胞(neutrophil)是在细胞水平研究其生物学特性及功能的基础。根据目前国内外文献资料显示的常用中性粒细胞分离方法,选取四种,即Percoll非连续密度梯度离心法、Ficoll-Hypaque密度梯度离心法、裂解红细胞法、Dextran作用下红细胞自然沉降法进行细胞纯度、回收率、存活率比较,旨在寻找一种简单、高效的中性粒细胞分离方法。方法:采取健康人外周静脉血,分别采用以上四种方法进行中性粒细胞分离,对分离纯化的中性粒细胞纯度、回收率、存活率进行比较。结果:Percoll非连续密度梯度离心法与Ficoll-Hypaque密度梯度离心法分离得到的细胞纯度均大于90%,两者间比较无显著差异(P＞0.05);裂解红细胞法和Dextran作用下红细胞自然沉降法分离得到的细胞纯度略低于Percoll非连续密度梯度离心法和Ficoll-Hypaque密度梯度离心法(P＜0.05或P＜0.01);Percoll非连续密度梯度离心法、Ficoll-Hypaque密度梯度离心法和裂解红细胞法的回收率均高于Dextran作用下红细胞自然沉降法(P＜0.01或P＜0.05);Percoll非连续密度梯度离心法回收的中性粒细胞存活率明显高于其它三组(P＜0.01或P＜0.05)。结论:Percoll非连续密度梯度离心法分离中性粒细胞,纯化程度好,回收率和细胞存活率高,是一种简单、高效的中性粒细胞分离方法,适于临床和科研中广泛应用。第二章AF-1对于内毒素诱导的中性粒细胞与内皮细胞粘附的影响及机制研究目的:中性粒细胞(neutrophil)在肺内聚集是急性肺损伤(acutelung injury,ALI)发病的最初环节,中性粒细胞与肺血管内皮细胞粘附是炎症反应的最初现象,是其进一步移行入肺组织的基础。子宫珠蛋白(uteroglobin,UG)是一种多功能非类固醇类激素蛋白,具有抗炎、抗氧化、免疫调节、抑制肿瘤发生等作用。来源于子宫珠蛋白C末端第三个α螺旋的九肽Antiflammin-1(AF-1),也被证实具有强烈的抗炎作用。关于AF-1抗炎作用的机制至今尚未完全明了,因此本研究旨在通过观察AF-1对于内毒素诱导的中性粒细胞与内皮细胞粘附及粘附分子表达的影响,检测中性粒细胞和内皮细胞子宫珠蛋白结合蛋白(Uteroglobin-binding protein,UGBP)的表达情况,通过应用制备的抗-UGBP抗体观察抗-UGBP抗体对AF-1粘附抑制及粘附分子表达的影响,还通过检测AF-1、抗-UGBP抗体对于P38/MAPK信号转导通路的影响,初步探讨AF-1产生抗炎作用的机制,为ALI的治疗提供新的靶点。方法:采取健康人外周静脉血分离纯化中性粒细胞,培养人脐静脉内皮细胞(HUVEC-12),分别应用不同浓度、不同作用时间的AF-1预处理中性粒细胞和内皮细胞,观察中性粒细胞与内皮细胞粘附率的变化及应用流式细胞仪检测粘附分子表达的变化;通过应用免疫荧光、流式细胞术、RT-PCR一系列实验技术检测中性粒细胞和内皮细胞UGBP的表达情况;应用制备的抗-UGBP抗体预处理观察抗-UGBP多抗对AF-1粘附抑制及粘附分子表达的影响;进一步应用Western-Blot技术检测AF-1、抗-UGBP抗体对于P38/MAPK信号转导通路的影响。结果:1.应用AF-1(100μmol/L)分别预处理内皮细胞、中性粒细胞15min～2h时可以明显抑制内毒素诱导的中性粒细胞与内皮细胞的粘附,与LPS组比较均有统计学意义(P＜0.01),其中以30min预处理抑制作用最明显,随着预处理时间延长,其粘附抑制效应均降低,预处理时间为4h时,其粘附抑制作用与LPS组比较没有统计学差异;不同浓度AF-1分别预处理内皮细胞、中性粒细胞30min,研究发现AF-1 1μmol/L对中性粒细胞与内皮细胞的粘附即产生抑制作用(P＜0.05),随着AF-1浓度增高其粘附抑制作用逐渐增强,呈剂量依赖性。流式细胞仪检测结果发现AF-1可以抑制内毒素诱导的中性粒细胞粘附分子CD11b和内皮细胞粘附分子CD54的表达,而AF-1本身对于静息状态下的中性粒细胞的CD11b和内皮细胞的CD54表达没有影响。2.免疫荧光、RT-PCR、流式细胞实验均证实在内皮细胞和中性粒细胞上均存在UGBP的表达,并且中性粒细胞的表达强于内皮细胞。3.粘附率测定及粘附分子表达测定结果显示抗-UGBP抗体预处理可以抑制AF-1的粘附抑制效应。4.进一步应用Western-Blot检测AF-1、抗-UGBP抗体对于P38/MAPK磷酸化水平的影响。结果显示,AF-1可以明显下调内毒素诱导的内皮细胞P38/MAPK的磷酸化水平,抗-UGBP抗体可以抑制AF-1作用引起的下调效应。结论:1.AF-1可以抑制内毒素诱导的中性粒细胞与内皮细胞的粘附及粘附分子表达,说明AF-1抗炎作用的产生与调节粘附分子的表达有关系。2.中性粒细胞和内皮细胞均有UGBP表达,以细胞膜表达最为强烈,细胞浆内也有少量表达,并且中性粒细胞UGBP的表达水平高于内皮细胞。3.抗-UGBP抗体预处理可以抑制AF-1对于粘附的抑制作用及粘附分子表达变化,说明AF-1的粘附抑制作用同UGBP密切相关。4.AF-1可以通过与UGBP结合调节细胞内P38/MAPK磷酸化水平而影响细胞内信号转导。因此我们认为AF-1通过结合细胞表面UGBP进而调节细胞内信号通路P38/MAPK的磷酸化水平而抑制中性粒细胞与内皮细胞的粘附及粘附分子的表达,产生抗炎作用。更多还原

【Abstract】 Chapter1 Comparison of different neutrophil separation methods from human peripheral bloodObjective:To study neutrophil’s biological characteristic and function,it is the key step to isolate neutrophils from the peripheral blood. Percoll density gradient centrifugation,Ficoll-Hypaque density gradient centrifugation,red blood cells cracking and the natural erythrocyte sedimentation method with Dextran are the four popular methods.The objective of this work was to investigate a simple and efficient method from the four methods.Methods:Respectively using the four methods to separate neutrophil from healthy human peripheral blood,then the purity,recovery rate,and cell viability were compared.Results:Among the four methods,the neutrophil purity from Percoll density gradient centrifugation and Ficoll-Hypaque density gradient centrifugation were more than 90%,but there was no significant difference between them(P＞0.05).The neutrophil recovery rate from Percoll density gradient centrifugation,Ficoll-Hypaque density gradient centrifugation and red blood cells cracking were significantly higher than that from the natural erythrocyte sedimentation method with Dextran(P＜0.01 or P＜0.05).Cell viability from Percoll density gradient centrifugation was significantly higher than those from others(P＜0.01 or P＜0.05)Conclusion:Percoll density gradient centrifugation provides the most simple and efficient approach for isolation of human blood neutrophil with the high purity and viability. Chapter2 The influence of Antiflammin-1 on adhension between neutrophils and endothelial cells induced by LPSObjective:The accumulation of neutrophil is the initial factor in the acute lung injury(ALI).The adhension between neutrophils and pulmonary vascular endothelial cells is the primary step in the inflammatory response,which is the basis for neutrophils migration into the lung.Uteroglobin(UG)is a steroid-inducible,homodimerie, multifunctional secreted protein with potent anti-inflammatory, immunomodulatory,anti-tumorigenic and embryonic growth-stimulatory properties.Antiflammin-1(MQMKKVLDS,AF-1)is equivalent to the 9 C-terminal amino acids ofα-helix 3 of uteroglobin and have been showed have potent anti-inflammatory effects.But the mechanism of AF-1 which inhibites the inflammation is still unclear.The present study was determined to investigate the effects of AF-1 on adhension between neutrophils and human umbilical vein endothelial cells(HUVECs)and on the expression of adhesion molecule on human neutrophil and HUVEC induced by LPS.Furthermore,we detected the expression of uteroglobin-binding protein(UGBP)on neutrophils and endothelial cells, and the adhesion inhibition of anti-UGBP antibody and the adhesion molecules expression induced by AF-1.We also detected the effect of AF-1 and anti-UGBP antibody on signal pathway of P38/MAPK to provide a new target of ALI treatment.Methods:We analyzed in vitro the adherence of neutrophils to HUVEC-12 cell line after pretreatment with AF-1 of different time and different concentration,and observed the expression of adhesion molecules on human neutrophils and HUVECs using the flow cytometry (FCM).Furthermore,we investigated the expression of UGBP on neutrophils and endothelial cells by Immunofluorescence,FCM and RT-PCR.And we observed the adhesion inhibition of anti-UGBP antibody and the adhesion molecules expression induced by AF-1 using the flow cytometry(FCM).Moreover,the effect of AF-1 and anti-UGBP antibody on the signal pathway of P38/MAPK was detected using Western-Blot.Results:1.Neutrophils adhension to endothelial cells were significantly inhibited compared with the LPS-stimulated group(P＜0.01),while neutrophils or endothelial cells were pretreated with AF-1(100μmol/L) for 15min～2h,and the inhibitory effect reached the maximum at 30min, with prolonging the pretreatment time its inhibitory effect decreased,for example,its inhibitory effect does not have statistics difference compared with LPS group at 4h.When nentrophils or endothelial cells were treated with different concentration of AF-1 from 1μmol/L to 100μmol/L for 30min,we found that AF-1(1μmol/L)could inhibited the adhesion between nentrophils and endothelial cells(P＜0.05),with a concentration-dependent relationship.FCM assay found that AF-1 could inhibit the expression of adhesion molecule CD11b on neutrophils and CD54 on endothelial cells induced by LPS,but the AF-1 itself does not affect the expression of adhesion molecule on the resting neutrophils and endothelial cells.2.Immunofluorescence,FCM assay and RT-PCR indicated that there is UGBP expression in both neutrophils and endothelial cells,and UGBP expression in nentrophils was higher than in endothelial cells.3.Anti-UGBP antibody pretreatment could attenuate the inhibition of adhesion rate and the expression of adhesion molecules induced by AF-1.4.Western-Blot assay indicated that AF-1 could reduce LPS-induced the phosphorylation level of P38/MAPK in endothelial cells significantly, and anti-UGBP antibody could inhibit the down-regulated effect of AF-1.Conclusions:1.AF-1 can inhibit the adhesion between neutrophils and endothelial cells and the expression of adhesion molecules induced by LPS,which suggests that the anti-inflammatory effect of AF-1 may be related to modulate adhesion molecules expression.2.UGBP is expressed in both neutrophils and endothelial cells,but UGBP expression in neutrophils was higher than in endothelial cells. UGBP receptor expression in membrane was higher than in cytoplasm. 3.The pretreatment by anti-UGBP antibody can attenuate the inhibitory effect of the adhesion between neutrophils and endothelial cells and the expression of adhesion molecules induced by AF-1,which suggest the inhibitory effect of AF-1 on adhesion might be related to the UGBP.4.The AF-1 can decrease the intracellular phosphorylation level of P38/MAPK by binding to UGBP.These results provided evidences that AF-1 can inhibit the adhesion between neutrophils and endothelial cells and the adhesion molecules expression on them to produce anti-inflammatory effect,which mechanism is related to decreasing the phosphorylation level of P38/MAPK through combination with the UGBP.更多还原