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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0.18mJ/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p38MAPK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p38MAPK,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。
BACKGROUND: Previous studies have found that shock wave enhances the proliferation and secretion of CD3 and CD28-stimulated T cells by activating mitogen-activated protein kinase p38 (p38MAPK). OBJECTIVE: To observe whether ATP release from the cells is a potential mechanism of shock wave enhancing T cell function. Design: The cells were observed, the group control repeated measurement observation. Unit: First Hospital of Jilin University, Department of Orthopedics. Material: KDE-2001 extracorporeal shock wave lithotripsy machine (Beijing Branch Jian'an Medical Technology Co., Ltd.). The phosphorylated p38 MAPK kit (Cell Signaling Tehchmology, Inc. USA); the P2 receptor inhibitor: suramin 50 mg (BIOMOL Research Laboratories Inc, PA) was treated with 1.7492 mL of MAPKp38 inhibitor (BioSource Inc., Camarillo, Of IMDM medium 50 mg of suramin is dubbed 0.02 mol / L solution. (BioSource Inc., USA * 542 Flynn Roas, Camarillo, CA); p38 MAPK inhibitor: SB2035801 mg ATP-based bioluminescence kit / ATP Bioluminescence Assay Kit CLS II (Roche Diagnostics GmbH, Mannheim, Germany). Methods: The experiment was performed in the Department of Orthopedics, the First Hospital of Jilin University from January 2005 to December 2006. ① The impact of shock wave lithotripsy (working voltage 7kV, capacitance 0.3μF, shock wave normal phase pressure 23MPa, energy density 0.18mJ / mm2) on T cells, the shock wave effect 50 to 400 times. ② using a specific ATP kit to detect low-energy shock wave caused by T cells (human peripheral blood mononuclear cells and Jurkat T cells) ATP secretion. ③ set no antagonist and inhibitor negative control group. Peripheral blood mononuclear cells were used to detect the impact of low-energy shock waves on the activation of T lymphocytes. Jurkat cells were used to detect the effect of low-energy shock waves on the secretion of interleukin-2 by T lymphocytes. Anti-p38MAPK antibody and anti-phosphorylated p38MAPK Thr180 / Tyr182) antibodies were used to determine p38MAPK expression and phosphorylation on Jurkat T cells by Western blotting. MAIN OUTCOME MEASURES: ATP content outside T cells, interleukin 2 content in cell suspension, cell proliferation and phosphorylation of p38 MAPK in cells. Results: ① At 100, 150, 200, 250, 300, 360 and 400 shocks, the content of extracellular ATP was significantly increased (P <0.01) and the content of ATP was dependent on the number of shock waves. (2) Phytohemagglutinin-activated peripheral blood mononuclear cells activated by apyrase, KN-62 and suramin or Jurkat T cells activated by CD3 and CD28 were treated with shock wave with an energy density of 0.18 mJ / mm2 for 100,150,200,250,300,330, -TdR incorporation was lower (P <0.01) than that of the negative control group without adding apyrase, KN-62 or suramin, and the content of cytokin-2 in the cell supernatant was significantly increased (P <0.01) . The level of p38MAPK induced by shock waves in Jurkat T cells was significantly reduced by the addition of ATP, KN-62 or suramin. Conclusion: (1) Low-energy shock wave can damage the cell membrane without damaging other organelles, leading to excessive secretion of ATP from T lymphocytes to extracellular space. Excess extracellular ATP activates P2X7 receptor and activates a large number of p38MAPK in cells Phosphorylation of p38MAPK as a costimulatory agent enhances the proliferation of activated T lymphocytes and secrete interleukin - 2. ② In low-energy shock wave on the regulation of T lymphocytes, T cells secrete ATP plays a very important role.