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
충격파통과ATP석방、P2수체급격활p38MAPK격매촉진T세포증식화분비백세포개소2
Shock waves co-stimulate T-cell proliferation and interleukin-2 expression through ATP release, P2 receptor and p38 mitogen activated protein kinase activation
  • 万方数据
    中国组织工程研究与临床康复 중국조직공정연구여림상강복
    JOURNAL OF CLINICAL REHABILITATIVE TISSUE ENGINEERING RESEARCH
    2007年 31期 6305-6310 ,共6页

    于铁成%赵毅%陈玮伦%金安%刘建国

    우철성%조의%진위륜%금안%류건국

    促分裂原活化蛋白激酶p38%白细胞介素2%T细胞增殖%ATP 促分裂原活化蛋白激酶p38%白細胞介素2%T細胞增殖%ATP
    촉분렬원활화단백격매p38%백세포개소2%T세포증식%ATP
    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:SB203580 1 mg(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:suramin 50 mg(BIOMOL Research Laboratories Inc,PA),用1.749 2 mL的IMDM培养基将50 mg的suramin配成0.02 mol/L的溶液.ATP酶:apyrase 200 U(Sigma,U.S.A.);P2X7受体阻滞剂:KN-62(BioSource Inc.,Camarillo,CA);p38 MAPK抑制剂:SB203580 1 mg(BioSource Inc.,USA*542 Flynn Roas,Camarillo,CA);检测ATP的生物荧光试剂盒/ATP Bioluminescence Assay Kit CLS Ⅱ(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7 kV、电容0.3μF时,冲击波正相压强23 MPa,能量密度0.18 mJ/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-p38 MAPK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p38 MAPK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的增加含量和冲击波的作用次数有依从关系.②加入apyrase,KN-62,suramin的植物血凝素激活的外周血单个核细胞细胞或CD3和CD28激活的Jurkat T细胞,在能量密度为0.18 mJ/mm2的冲击波作用100,150,200,250,300,330次时,细胞对3H-TdR掺入量比没有加入apyrase、KN-62或suramin的阴性对照组低(P＜0.01),细胞上清液中的的细胞介素-2的活性含量表现为明显增高(P＜0.01).加入ATP、KN-62或suramin后,冲击波激活Jurkat T细胞的p38 MAPK的程度明显降低.结论:①低能冲击波能损伤细胞膜而不损伤其他细胞器,引起T淋巴细胞内的ATP过多向细胞外分泌,细胞外过量的ATP过多地激活了P2X7受体,激活细胞内的大量的p38 MAPK,最后磷酸化的p38MAPK作为协同刺激因子增强激活的T淋巴细胞增殖及分泌白细胞介素2.②在低能冲击波对T淋巴细胞的功能调节上,T细胞分泌的ATP起到非常重要的作用.
    배경:이왕연구발현,충격파가통과격활유사분렬원격활단백격매p38(p38MAPK),증강CD3화CD28격활적T세포증식화분비백세포개소2.목적:관찰세포내적ATP향외석방,시부위충격파증강T세포공능적잠재궤제.설계:이세포위관찰대상,분조대조중복측량관찰.단위:길림대학제일의원골과연구실.재료:KDE-2001체외충격파쇄석궤(북경중과건안의료기술공사제조).MAPKp38억제제:SB203580 1 mg(BioSource Inc.,Camarillo,CA);검측린산화적p38MAPK시제합(Cell Signaling Tehchmology,Inc.U.S.A.);P2수체억제제:suramin 50 mg(BIOMOL Research Laboratories Inc,PA),용1.749 2 mL적IMDM배양기장50 mg적suramin배성0.02 mol/L적용액.ATP매:apyrase 200 U(Sigma,U.S.A.);P2X7수체조체제:KN-62(BioSource Inc.,Camarillo,CA);p38 MAPK억제제:SB203580 1 mg(BioSource Inc.,USA*542 Flynn Roas,Camarillo,CA);검측ATP적생물형광시제합/ATP Bioluminescence Assay Kit CLS Ⅱ(Roche Diagnostics GmbH,Mannheim,Germany).방법:실험우2005-01/2006-12재길림대학제일의원골과연구실완성.①채용체외충격파쇄석궤(공작전압7 kV、전용0.3μF시,충격파정상압강23 MPa,능량밀도0.18 mJ/mm2)작용우T세포,충격파작용50～400차.②용특이성적ATP시제합검측저능충격파시부인기T세포(인외주혈단개핵세포화Jurkat T세포)분비ATP.③설무길항제화억제제적음성대조조.용인외주혈단개핵세포검측저능충격파대격활적T림파세포증식적영향,용Jurkat세포검측저능충격파대T림파세포분비백세포개소2적영향,용항-p38 MAPK항체급항-린산화적p38MAPK(Thr180/Tyr182)항체통과면역인적법측정Jurkat T세포상적p38 MAPK적표체급린산화.주요관찰지표:T세포외적ATP함량、세포현액중적백세포개소2적함량、세포적증식화세포p38MAPK적린산화정도.결과:①충격파작용100,150,200,250,300,360화400차시교무충격파작용시세포외적ATP적함량명현증가(P＜0.01),병차ATP적증가함량화충격파적작용차수유의종관계.②가입apyrase,KN-62,suramin적식물혈응소격활적외주혈단개핵세포세포혹CD3화CD28격활적Jurkat T세포,재능량밀도위0.18 mJ/mm2적충격파작용100,150,200,250,300,330차시,세포대3H-TdR참입량비몰유가입apyrase、KN-62혹suramin적음성대조조저(P＜0.01),세포상청액중적적세포개소-2적활성함량표현위명현증고(P＜0.01).가입ATP、KN-62혹suramin후,충격파격활Jurkat T세포적p38 MAPK적정도명현강저.결론:①저능충격파능손상세포막이불손상기타세포기,인기T림파세포내적ATP과다향세포외분비,세포외과량적ATP과다지격활료P2X7수체,격활세포내적대량적p38 MAPK,최후린산화적p38MAPK작위협동자격인자증강격활적T림파세포증식급분비백세포개소2.②재저능충격파대T림파세포적공능조절상,T세포분비적ATP기도비상중요적작용.
    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/mm2 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 Jurkat T-cell were measured by Western Immunoblotting with anti-p38 MAPK antibodies and anti-p38 MAPK phospho-specific antibodies that recognized the phosphorylation (on Thr180/Tyr182).MAIN OUTCOME MEASURES: extra-cellular ATP release, IL-2 expression in cell suspension, cellular proliferation and the phosphorylation of p38 MAPK.RESULTS: ①ATP release under the condition without LDSWs was obviously lower than that with LDSWs of 100, 150,200, 250, 300, 360 and 400 impulses (P ＜ 0.01), and ATP release increased with the LDSWs impulse.②Compared with negative control group, the additions of apyrase, KN-62 or suramin attenuated the 3H-TdR incorporation of the phytohemagglutinin-stimulated PBMCs or CD3/CD28-stimulated Jurkat T-cells, which were effected with LDSWs of 100,150, 200, 250, 300, 330 impulses at 0.18 mJ/mm2 (P＜ 0.01). IL-2 expression in the cellular supernatant was also significant increased (P ＜ 0.01). ATPase, KN-62 or suramin all decreased the effect of LDSWs on p38 MAPK of Jurkat T-cells.CONCLUSION: ①LDSWs deform cellular membranes but have no effect on organelle, which results in ATP release from Jurkat cells. Exogenous ATP release activates P2X7 receptor and p38 MAPK, and increases IL-2 expression. LDSWs enhance T-cell proliferation and IL-2 expression through a mechanism that involves ATP release, P2X7 receptor and phosphorylized p38 MAPK activation. ②The release of ATP plays a key role in the mechanism through which LDSWs regulate the function of T cells.
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