STIM1抗体封闭对fMLP诱导的人中性粒细胞极性及趋化性的影响

发布时间：2018-01-08 14:08

本文关键词：STIM1抗体封闭对fMLP诱导的人中性粒细胞极性及趋化性的影响　出处：《南方医科大学》2011年硕士论文　论文类型：学位论文

更多相关文章： STIM1 fMLP 中性粒细胞 极性 趋化性

【摘要】：研究背景中性粒细胞是先天免疫系统的重要组成部分。当机体遭受外来细菌和病原体入侵时,充当第一道防线,它们的主要功能是渗出血管壁并迁移到炎症灶,通过吞噬作用和释放超氧化物和／或蛋白水解酶杀死细菌和入侵的微生物。这种外渗和迁移的机制是通过细胞的极性和趋化性来实现的。临床上一些疾病例如败血症、哮喘、缺血／再灌注损伤和器官移植的排斥反应、动脉粥样硬化、病毒性心肌炎、类风湿性关节炎、过敏反应、炎症性皮肤病甚至肿瘤的发生和转移都与中性粒细胞极性功能的增强和减弱有关。因此深入探讨中性粒细胞极性调节机制为抵抗中性粒细胞激活导致的机体组织损伤寻找到新的干预和治疗靶点。钙信号调控细胞内许多重要生理、病理过程,胞质内Ca2+浓度升高依赖于两种途径：内质网钙库释放和细胞膜外钙内流。钙池操纵的钙流入(store-operated calcium entry, SOCE)是非兴奋细胞的主要钙流入形式。近年来随着蛋白基质交互分子(stromal interaction molecule,STIM)的发现,将SOCE调控机制的研究向前推进了一步。Liou, Roos等证实了STIM1而非STIM2调控着SOCE。STIMl作为SOCE的关键分子,主要存在于内质网(Endoplasmic Reticulum, ER)内,也有少量分布于质膜(plasma membrane,PM)上,它包含着一个定位于ER内的EF-hand基序,充当着钙离子感受器的角色。文献报导SOCE在细胞极性及趋化过程中发挥着重要作用,但对于STIM1分子在其中的作用及作用机制缺乏研究。细胞的极性机制研究中存在着依赖PI3K和非依赖PI3K两条途径。PI3K-Akt被认为是经典的极性信号通路,也有研究表明酪氨酸激酶Src、Rho小G蛋白参与极性形成、细胞的迁移、肿瘤的侵袭与转移等。而对于fMLP诱导的人中性粒细胞极性及趋化过程中是否也存在这样的信号传导通路尚不清楚。人中性粒细胞为终末血细胞,在体外存活时间短,难于进行基因操作或RNA干扰,那么电转抗体封闭技术对于这种短时程的研究不失为一种行之有效的手段。为了阐明SOCE在人中性粒细胞极性及趋化性的作用以及调控机制,本实验采用此技术封闭细胞内STIM1蛋白分子,观察其对中性粒细胞趋化率的影响,以及相关的信号蛋白分子Akt, Src, Rho小G蛋白的变化。目的本实验通过研究STIM1抗体封闭后对fMLP诱导的人中性粒细胞极性及趋化性的影响,探讨SOCE在人中性粒细胞极性及趋化性中的作用及其调控机制。方法 1.采用Dextran作用下红细胞自然沉降,Ficoll-Hypaque密度梯度离心,低渗裂解红细胞的方法急性分离人外周血中性粒细胞。 2.使用电转仪将anti-STIM1抗体转入人中性粒细胞内以进行后续的人中性粒细胞极性及趋化性的研究。 3.采用Immunoprecipitation和Western blotting检测anti-STIM1抗体电转效果。 4.使用Zigmond chamber观察STIM1抗体封闭后fMLP诱导的人中性粒细胞趋化率的变化。 5.应用GST pull-down and Western blotting方法检测STIM1抗体封闭后fMLP诱导的人中性粒细胞极性及趋化过程中相关信号分子的变化。结果 1. Immunoprecipitation和Western blotting检测anti-STIM1抗体电转效率显著,电转anti-STIM1抗体组检测到的蛋白信号强于非电转组(P0.001)。 2. Control组、IgG组、anti-STIM1三组间趋化率比较差异有统计学意义(P0.001),趋化率的大小为Control组IgG组anti-STIM1组。 3.免疫印迹结果显示Control组加与不加fMLP刺激p-Akt检测到的蛋白条带信号很弱,差异无统计学意义(P=0.505), Src磷酸化以及Racl,Rac2,Cdc42活化水平加fMLP刺激后高于不加fMLP(P0.001); IgG组有着相同趋势,p-Akt无变化(P=0.766), p-Src,GTP-Racl,GTP-Rac2,GTP-Cdc42在fMLP刺激后其含量高于不加fMLP(P0.001); anti-STIM1组p-Akt差异无统计学意义(P=0.476), p-Src (P=0.315), GTP-Racl (P=0.944), GTP-Rac2 (P=0.386), GTP-Cdc42 (P=0.386)加与不加fMLP刺激各组比较差异均无统计学意义。结论 1. STIM1抗体封闭后抑制了fMLP诱导的人中性粒细胞极性及趋化性,提示SOCE可能参与了fMLP诱导的人中性粒细胞极性和趋化性。 2. STIM1抗体封闭后下调Src和Rac/Cdc42的活性,说明SOCE通过Src-Rac/Cdc42信号通路正性调节fMLP诱导的人中性粒细胞极性及趋化性。
[Abstract]:Background of the study Neutrophils are an important part of innate immune system . When the organism is invaded by foreign bacteria and pathogens , it acts as the first line of defense . Their main function is to exude the blood vessel wall and migrate to the inflammatory range . The mechanism of this extravasation and migration is achieved by the polarity and the convergence of the cells . The mechanism of this extravasation and migration is related to the enhancement and attenuation of the neutrophil function by phagocytosis and release of superoxide and / or proteolytic enzymes . There are many important physiological and pathological processes in calcium signal regulation cells . The increase of intracellular Ca 2 + concentration depends on two ways : calcium influx in endoplasmic reticulum and intracellular calcium influx . In recent years , the study of SOCE control mechanism has been advanced by one step . In recent years , as the key molecule of SOCE , it contains an EF - hand motif located in ER , which acts as a role of calcium ion receptor . The literature reports that SOCE plays an important role in the process of cell polarity and chemotropism . In the study of the polarity mechanism of the cells , there are two pathways , which are dependent on the K 3 + and the non - dependent kinase , which are considered to be classical polar signal pathways . It is also found that there is a clear signal transduction pathway in the process of human neutrophil polarity and chemotropism induced by fmlp . In order to clarify the effect of SOCE on human neutrophil polarity and chemotropism and the mechanism of regulation and regulation , this experiment used this technique to close the molecule of STIM1 protein in the cell and observe its effect on neutrophil chemoattractant , as well as the changes of the related signal protein molecules such as signal protein kinase , protein kinase and protein . Purpose The effects of SOCE on the polarity and chemokine of human neutrophil in human neutrophil and its mechanism were investigated by studying the effect of STIM1 antibody on the polarity and chemoattractant of human neutrophil in human neutrophils . method 1 . Human peripheral blood neutrophils were isolated from human peripheral blood by the method of natural sedimentation of red blood cells , Ficoll - Hyacinth density gradient centrifugation and hypotonic lysis of red blood cells . 2 . The anti - STIM1 antibody was transferred to human neutrophils using an electrometer to conduct subsequent studies on the polarity and chemokine of human neutrophils . 3 . The effect of anti - STIM1 antibody was detected by Immunoppressing and Western blotting . 4 . Observe the change of human neutrophil chemoattractant induced by the STIM1 antibody after closure of the STIM1 antibody . 5 . GST pull - down and Western blotting were used to detect the changes of related signal molecules in the human neutrophil polarity and chemoattractant induced by STIM1 antibody . Results 1 . Immunoperoxidase and Western blotting showed that the anti - STIM1 antibody was significantly more efficient , and the protein signal detected by anti - STIM1 antibody group was stronger than that of non - electric rotating group ( P0.001 ) . 2 . There was significant difference between the three groups of control group , IgG group and anti - STIM1 group ( P0.001 ) , and the size of Chemotactic rate was control group IgG group anti - STIM1 group . 3 . There was no statistical significance ( P = 0.505 ) , there was no significant difference ( P = 0.505 ) , no change ( P = 0.766 ) , p - src ( P = 0.315 ) , GTP - Racl ( P = 0.944 ) , GTP - Rac2 ( P = 0.386 ) , GTP - Cdc42 ( P = 0.396 ) , GTP - Cdc42 ( P = 0.394 ) , GTP - Rac2 ( P = 0.386 ) , GTP - Cdc42 ( P = 0.394 ) , GTP - Rac2 ( P = 0.386 ) , GTP - Cdc42 ( P = 0.394 ) , GTP - Rac2 ( P = 0.386 ) , GTP - Cdc42 ( P = 0.394 ) , GTP - Rac2 ( P = 0.386 ) , GTP - Cdc42 ( P = 0.394 ) , GTP - Rac2 ( P = 0.386 ) , GTP - Cdc42 ( P = 0.394 ) , GTP - Rac2 ( P = 0.386 ) , GTP - Cdc42 ( P = 0.394 ) , GTP - Rac2 ( P = 0.386 ) , GTP - Cdc42 ( P = 0.394 ) , GTP - Rac2 ( P = 0.386 ) , GTP - Cdc42 ( P = 0.394 ) , GTP - Rac2 ( P = 0.386 ) , GTP - Cdc42 ( P = 0.394 ) , GTP - Rac2 ( P = 0.386 ) , GTP - Cdc42 ( P = 0.394 ) , GTP - Rac2 ( P = 0.386 ) , GTP - Cdc42 ( P = 0.394 ) , GTP - Rac2 ( P = 0.386 ) , GTP - Cdc42 ( P = 0.394 ) , GTP - Rac2 ( P = 0.386 ) , GTP - Cdc42 ( P = 0.394 ) , GTP - Rac2 ( P = 0.386 ) , GTP - Cdc42 ( P = 0.394 ) , GTP - Rac2 ( P = 0.386 ) , GTP - Cdc42 ( P = 0.394 ) , GTP - Rac2 ( P = 0.386 ) , GTP - Cdc42 ( P = 0.394 ) , GTP - Rac2 ( P = 0.386 ) , GTP - Cdc42 ( P = 0.386 ) , and no statistical significance . P=0.4 1 . After the blocking of the STIM1 antibody , the human neutrophil polarity and the chemoattractant induced by fmlp were inhibited , suggesting that SOCE might be involved in the human neutrophil polarity and chemokine induced by fmlp . 2 . After the STIM1 antibody was closed down , the activity of c / c / cdc42 was downregulated , indicating that SOC EON was positive to regulate the polarity and chemokine of human neutrophils induced by fL - Rac / Cdc42 signal pathway .

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

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