冲击波通过ATP释放、P2受体及激活p38MAPK激酶促进T细胞增殖和分泌白细胞介素2(英文)  

Shock waves co-stimulate T-cell proliferation and interleukin-2 expression through ATP release, P2 receptor and p38 mitogen activated protein kinase activation

作　　者：于铁成[1] 赵毅[1] 陈玮伦[2] 金安[1] 刘建国[1] 

机构地区：[1]吉林大学第一医院骨科,吉林省长春市130021 [2]吉林大学第一医院耳鼻喉科,吉林省长春市130021

出　　处：《中国组织工程研究与临床康复》2007年第31期6305-6310,共6页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金资助项目(30500132)~~

摘　　要：背景:以往研究发现,冲击波可通过激活有丝分裂原激活蛋白激酶p38(p38MAPK),增强CD3和CD28激活的T细胞增殖和分泌白细胞介素2。目的:观察细胞内的ATP向外释放,是否为冲击波增强T细胞功能的潜在机制。设计:以细胞为观察对象,分组对照重复测量观察。单位:吉林大学第一医院骨科研究室。材料:KDE-2001体外冲击波碎石机(北京中科建安医疗技术公司制造)。MAPKp38抑制剂:SB2035801mg(BioSource Inc.,Camarillo,CA);检测磷酸化的p38MAPK试剂盒(Cell Signaling Tehchmology,Inc.U.S.A.);P2受体抑制剂:suramin50mg(BIOMOL ResearchLaboratories Inc,PA),用1.7492mL的IMDM培养基将50mg的suramin配成0.02mol/L的溶液。ATP酶:apyrase200U(Sigma,U.S.A.);P2X7受体阻滞剂:KN-62(BioSource Inc.,Camarillo,CA);p38MAPK抑制剂:SB2035801mg(BioSource Inc.,USA*542Flynn Roas,Camarillo,CA);检测ATP的生物荧光试剂盒/ATP BioluminescenceAssay Kit CLSⅡ(Roche Diagnostics GmbH,Mannheim,Germany)。方法:实验于2005-01/2006-12在吉林大学第一医院骨科研究室完成。①采用体外冲击波碎石机(工作电压7kV、电容0.3μF时,冲击波正相压强23MPa,能量密度0.18mJ/mm2)作用于T细胞,冲击波作用50￣400次。②用特异性的ATP试剂盒检测低能冲击波是否引起T细胞(人外周血单个核细胞和Jurkat T细胞)分泌ATP。③设无拮抗剂和抑制剂的阴性对照组。用人外周血单个核细胞检测低能冲击波对激活的T淋巴细胞增殖的影响,用Jurkat细胞检测低能冲击波对T淋巴细胞分泌白细胞介素2的影响,用抗-p38MAPK抗体及抗-磷酸化的p38MAPK(Thr180/Tyr182)抗体通过免疫印迹法测定Jurkat T细胞上的p38MAPK的表达及磷酸化。主要观察指标:T细胞外的ATP含量、细胞悬液中的白细胞介素2的含量、细胞的增殖和细胞p38MAPK的磷酸化程度。结果:①冲击波作用100,150,200,250,300,360和400次时较无冲击波作用时细胞外的ATP的含量明显增加(P<0.01),并且ATP的增加含量和BACKGROUND：The previous researches indicate that, shock waves can enhance the proliferation of T-cells and the expression of interleukin （IL）-2 through a mechanism that involves p38 mitogen activated protein kinase （MAPK）activation. OBJECTIVE： To investigate if adenosine triphosphate （ATP） release is an underlying mechanism through which low-density shock waves （LDSWs） augment T-cell function. DESIGN： Controlled repetitive measurement by groups, taking cells as subject. SETTING： Department of Orthopedics, the First Hospital of Jilin University. MATERIALS： KDE-2001 Extracorporeal Shockwave Lithotripter （Beijing Zhongke Jian An Meditechs Co., Beijing, China）.p38 MAPK inhibitor SB203580 1 mg （BioSource Inc., Camarillo, CA）; p38 MAPK kit for detecting phosphorylation （Cell Signaling Technology, Inc. U.S.A.）; P2 receptor inhibitor suramin 50 mg （BIOMOL Research Laboratories Inc., PA） was prepared into 0.02 mol/L solution by 1.749 2 mL IMDM. ATP enzyme： apyrase 200 U （Sigma, U.S.A.）; P2X7 receptor antagonist KN-62 （BioSource Inc., Camarillo, CA）; ATP Bioluminescence Assay Kit CLS Ⅱ （Roche Diagnostics GmbH,Mannheim, Germany）. METHODS： The experiment was carried out in the Orthopedic Laboratory of the First Clinical Hospital in Jilin University from January 2005 to December 2006. ①An Extracorporeal Shockwave Lithotripter （at 7 kV generator voltage, 0.3 μF capacitance, 23 MPa positive pressure, 0.18 mJ/mm^2 energy flux density） was applied for LDSWs treatment ranging from 50 to 400 impulses. ②ATP release into the culture supernatant from Jurkat T-cells or human peripheral blood mononuclear cells （PBMCs） was determined with a specific ATP Bioluminescence Assay Kit. ③Negative control group excluded antagonist or inhibitor. Human PBMCs were used to determine the effect of LDSWs on activated T-lymphocyte proliferation. Human Jurkat cells were used to study the effects of LDSWs on IL-2 expression. Expression and phosphorylation of p38 MAPK in Jurka

关 键 词：促分裂原活化蛋白激酶p38 白细胞介素2 T细胞增殖 ATP 

分 类 号：R318[医药卫生—生物医学工程]