甲基-β-环糊精对中性粒细胞极性化及其与细胞内游离钙离子浓度变化的关系  

Effect of methyl-beta-cyclodextrin on neutrophil polarization and its correlation to changes in cytosolic free Ca^(2+) concentration

作　　者：袁春华[1] 蔡春青[2] 王斌[1] 邹飞[2] 

机构地区：[1]江西科技师范学院,江西省南昌市330013 [2]南方医科大学公共卫生与热带医学学院,广东省广州市510515

出　　处：《中国组织工程研究与临床康复》2008年第29期5619-5622,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金资助项目(30672353)~~

摘　　要：背景:脂筏是质膜上富含胆固醇和鞘磷脂的微结构域。甲基-β-环糊精(Methyl-β-cyclodextrin,mβCD)为脂筏破裂剂,破坏膜脂的完整性。目的:观察mβCD对甲酰甲硫氨酰-亮氨酰-苯丙氨酸(N-formyl-methionyl-leucyl-phenylalanine,fMLP)诱导的中性粒细胞极性化过程中的作用,及其与胞内游离钙离子浓度(Cytosolic free Ca2+concentration,[Ca2+]i)的关系。设计、时间及地点:开放性实验,于2007-02在南方医科大学公共卫生与热带医学学院环境医学研究室完成。材料:fMLP由美国Phoenix pharmaceuticals公司提供,mβCD由美国Sigma公司提供。方法:取健康志愿者外周静脉血5mL,采用重复梯度密度离心的方法分离中性粒细胞。根据诱导中性粒细胞极性化处理条件的不同分为3组,正常对照组、fMLP组、mβCD+fMLP组。正常对照组不加入任何试剂。fMLP组加入100nmol/LfMLP孵育15min。mβCD+fMLP组先加入10mmol/LmβCD预孵育15min,然后加入100nmol/LfMLP孵育15min。最后3组均滴入终浓度为10μmol/Lfuro-3/AM避光孵育45~60min。主要观察指标:倒置显微镜下观察中性粒细胞在不同处理条件下的细胞极性化情况,使用激光共聚焦显微镜监测[Ca2+]i在mβCD抑制细胞极性化过程中的变化。结果:fMLP可以诱导中性粒细胞的极性化,而预先使用10mmol/LmβCD孵育15min后,fMLP诱导的中性粒细胞极性化受抑制。同时,极性化过程中中性粒细胞的[Ca2+]i急剧增加,而mβCD预孵育后[Ca2+]i上升相幅度明显下降。结论:脂筏破裂剂mβCD抑制了fMLP诱导中性粒细胞极性化过程,该作用可能与[Ca2+]i变化有关。BACKGROUND： Lipid raft is a microarchitecture domain on the plasma membrane, with abundant of cholesterol and sphingomyelin. Methyl- β -cyclodextrin （m β CD） is a lipid raft Expansive Cracking Agent, OBJECTIVE： To investigate the effect of m β CD on neutrophil polarization induced by N-formyl-methionyl-leucyl-phenylalanine （fMLP） and its relationship with changes in cytosolic free Ca^2＋ concentration （[Ca^2＋]i.）. DESIGN, TIME AND SETTING： The opening experiment was performed at the Department of Environmental Medicine, College of Public Health and Tropical Medicine, Southern Medical University in February 2007. MATERIALS： fMLP and m β CD were respectively purchased from Phoenix pharmaceuticals and Sigma, USA. METHODS： 5 mL peripheral vein blood was collected from healthy volunteers. Neutrophils were separated by a discontinuous gradient density centrifugalization. According to different polarity disposal conditions, neutrophils were divided into 3 groups, normal control group, fMLP group and m β CD＋fMLP group. Neutrophils in the normal control group were intact. Neutrophils in the fMLP group were incubated with 100 nmol/L fMLP for 15 minutes. Neutrophils in the m β CD＋fMLP group were incubated with l0 mmol/L m β CD for 15 minutes, and then with 100 nmol/L fMLP for 15 minutes. Neutrophils in each group were incubated with 10 μ mol/L furo-3/AM for 45-60 minutes in the dark. MAIN OUTCOME MEASURES： The cell polarization was observed with an inverted microscope. The [Ca^2＋]i were measured using laser scanning confocal microscope during m β CD inhibits cell polarization. RESULTS： The fMLP induced the polarization of neutrophil. However, 10mM m β CD inhibited the formation of neutrophil polarity and the increasing of [Ca^2＋]i induced by fMLP. After m β CD incubation, increased [Ca^2＋]i levels significantly decreased. CONCLUSION： The m β CD inhibits the development of fMLP induced neutrophil polarity. This mechanism may be associated with [Ca^2＋]i changes.

关 键 词：中性粒细胞 极性化 [CA^2+]I mβCD 

分 类 号：R394.2[医药卫生—医学遗传学]