【作者】 于铁成；

【导师】 徐莘香；

【作者基本信息】 吉林大学 ， 骨外科学， 2004， 博士

【摘要】 冲击波是压力急剧变化的产物，能在几纳秒内达到它的压力最大值。冲击波的负相波段可引起空化反应。冲击波产生的快速上升的正相压力波和空化反应引起的微泡爆炸喷射会促使被作用物体表面的张力增加。一定的张力作用可使物体发生破裂。由于其这一特性，长期以来，高能冲击波一直被用于体外治疗泌尿系统的结石，并且取得了良好的疗效。近年来人们发现，低能冲击波对骨折愈合有促进作用，并且将冲击波应用到骨不连的治疗中，结果显示冲击波对肥大型骨不连治愈率几乎达到100%，对营养不良型骨不连的疗效不理想，这和手术治疗的结果相似。同时欧洲人又将冲击波用于慢性肌腱炎的治疗，如治疗肱骨外上髁炎、足底筋膜炎和钙化性肌腱炎等，也取得了理想疗效，与传统的非手术疗法相比，冲击波疗效更巩固，和手术治疗相比没有统计学上的差异。这样关于低能冲击波的应有基础研究开始展开。早在1986年Haupt就发现低能冲击波可促进小猪的创口愈合，而加大冲击波的强度则会抑制创口的愈合；形态学上的观察显示：低能冲击波会使小猪创口内的毛细血管、新生上皮细胞和巨嗜细胞的数量明显增加，是对照组的二倍。中国台北的Wang发现，能损伤细胞膜但不损伤其它细胞器的低能冲击波可诱导人骨髓间质干细胞向骨原细胞分化并诱导骨小节形成。那么，为什么低能冲击波能产生如此的生物学效应呢？对细胞产生一定应力和应变的机械刺激，会引起细胞内的ATP快速释放。一旦ATP被释放到细胞外的环境中，其会与表达在几乎所用哺乳动物细胞膜表面的P2X和/或P2Y受体相互作用，激活细胞内的传导路途经，调节细胞的功能和代谢。同时ATP的代谢产物（腺苷adenosine）也和细胞表面的P1受体相互作用。这样细胞外的ATP和其代谢产物通过它们的受体，对T淋巴细胞的功能产生很强的影响：ATP可促使小鼠的胸腺细胞增殖，ATP和腺苷将会抑制/或增强由T淋巴细胞受体激活的信号传导、细胞凋亡或胸腺细胞分化。冲击波会对细胞表面产生一定的应力和应变的机械刺激。如果能损伤细胞膜而不损伤其它细胞器的低能冲击波作用T淋巴细胞，将会产生什么样的影响呢? 该低能冲击波能否引起细胞内的ATP快速释放？释<WP=99>放到细胞外的ATP是否通过激活P2受体，引起相应的生物学效应呢？关于这方面的研究，国内外未见报道。另外，有丝分裂原激活蛋白激酶p38 （p38 MAPK）结构上和酵母菌的压力敏感蛋白HOG-1（the products of Saccharomyces cerevisiae osmosensing gene)蛋白相似。在受到高渗透压作用时，酵母菌细胞通过HOG-1蛋白调节基因表达。高渗透压或机械刺激作用可使人T淋巴细胞的p38 MAPK被激活，促进T淋巴细胞分泌IL-2。这样我们不禁要问：是否低能冲击波通过激活 p38 MAPK，对T淋巴细胞的功能产生影响？关于这方面的研究，国内外未见报道。为了检验这一假说，我们用Balb/c小鼠的脾细胞和人外周血单个核细胞（PBMC）来研究低能冲击波对T淋巴细胞增殖的影响。用人外周血单个核细胞（PBMC）和人Jurkat T细胞来研究低能冲击波对T淋巴细胞的信号传导和分泌IL-2的影响。研究目的：研究低能冲击波是否促进T淋巴细胞增殖及分泌IL-2；研究低能冲击波是否通过激活细胞内的有丝分裂原激活蛋白激酶p38（p38 MAPK）增强激活的T淋巴细胞增殖及分泌IL-2；探讨是否细胞内ATP向外释放是低能冲击波影响T淋巴细胞的信号传导和功能的潜在机理。材料和方法：研究低能冲击波是否能促进T淋巴细胞增殖及分泌IL-2先将Balb/c小鼠脾细胞、PBMC或Jurkat T细胞，用0.18mJ/mm2的低能冲击波分别作用0、100、150、200、250、300、320、360和420次。之后再立即用刀豆蛋白A(ConA)的亚刺激量作用Balb/c小鼠脾细胞，植物血凝素（PHA)的亚刺激量作用PBMC，或抗-CD3及抗-CD28抗体的亚刺激量作用Jurkat T细胞，同时设阴性对照组。继续放入5%CO2,37℃孵箱，继续培养48小时，用3H-TdR掺入法检测低能冲击波对激活的T淋巴细胞增殖的影响；或继续培养24小时，用生物活性分析法通过CTLL-2细胞检测低能冲击波对T淋巴细胞分泌IL-2的影响。研究p38 MAPK在低能冲击波促进T淋巴细胞的增殖和分泌IL-2的作<WP=100>用预先用p38 MAPK抑制剂( SB203580/20uM)和PBMC或Jurkat细胞37°C孵育箱共育1小时，再用冲击波和PHA或抗-CD3/抗-CD28抗体的亚刺激量作用，之后48小时用PBMC检测低能冲击波对激活的T淋巴细胞增殖的影响，或24小时用Jurkat细胞检测低能冲击波对T淋巴细胞分泌白细胞介素-2(IL-2)的影响。设无上述拮抗剂和抑制剂的阴性对照组。通过用免疫印迹法（Western Immunoblotting），用抗- p38MAPK抗体及抗-磷酸化的p38MAPK(Thr180/Tyr182)抗体，测定Jurkat T细胞上的p38 MAPK的表达及磷酸化。研究是否细胞内ATP向外释放是低能冲击波影响T淋巴细胞的信号传导和功能的潜在机理用人Jurkat T细胞通过ATP荧光试剂盒来检测低能冲击波是否引起T细胞分泌ATP。预先用P2X7受体的拮抗剂（KN-62/0.1uM）、P2受体的拮抗剂(suramin/200uM)、p38MAPK抑制剂( SB203580/0-50uM)或Ca2+螯合剂(EGTA/2mM) 和PBMC或Jurkat细胞37°C孵育箱共育1小时，之后用PBMC检测低能冲击波对激活的更多还原

【Abstract】 A shock wave is elicited by a transient pressure disturbance. Shock waves are characterized by high positive pressure, a rise time lower than 10 ns and tensile wave. The positive pressure and the short rise time are responsible for the direct shock wave effect and the tensile wave for the cavitation, which is called the indirect effect. The fast pressure transition of shock waves (high pressure, short rise time) cause very high tension at the exposed material surfaces so that the structure of the material cracks. Because of this, extracorporeal-generated shock waves were introduced approximately 20 years ago to disintegrate kidney stones. This treatment methed substantially changed the treatment of urolithiasis. Urology, however, is not the medical field for the potential use of shock waves for problems. Shock waves subsequently have been used in orthopaedics and traumatology to treat various insertional tendinopathies (enthesiopathies) and delayed unions and nonunions of fracture. Extracorporeal shock wave therapy has gained increasing acceptance in Europe for some musculoskeletal problems and has led to the inception of clinical studies in the United States. The subsequent researches on biologic mechanism of shock wave treatment on the orthopaedic disorders were carried on in the world. Haupt and Chvapil studies showed that low-density shock wave treatment (10 shock waves at 14kV) led to significant enhancement of reepithelialization of partial-thickness wounds in piglets. Histologically, the upper dermis in the animals that received the treatment had increased numbers of dilated microvessels and increased macrophages in the perivascular spaces. Also, the newly formed epithelial layer was four to five cells thick, almost twice as thick as in control wounds. Wang et al demonstrated that the low-density shock waves (an optimal dose of 500-impulse shock wave treatment at 0.16mJ/mm2) caused a rapid membrane hyperpolarization in 5 min, activation of Ras in 30min, production of specific osteogenic transcription factor (CBFA1) in 6h, enhancement of osteogenic growth factor (TGF-β1) production in 24 h (differentiation of bone marrow stromal cells toward osteoprogenitor associated with induction of <WP=104>TGF-β1), cell proliferation in 2 days, increase of bone alkaline phosphatase activation and collagen type I mRNA expression in 6 days, and osteocalcin mRNA expression in 12 days. Then why can the low-density shock waves (LDSWs) elicit such biological effects on cells? Mechanical stimulation can cause the rapid release of ATP from intracellular plasma. Once released into the extracellular environment, ATP can regulate cell function in an autocrine/paracrine manner by interacting with P2X and /or P2Y receptors that are expressed on the surface of virtually all mammalian cells. Extracellular ATP and its metablic products, including adenosine, which acts via P1 receptors, exert a strong influence on lymphocyte function: ATP can stimulate the proliferation of mouse thymocytes, and ATP and adenosine can antagonize and/or complement T cell receptor-induces signaling, apoptosis, and thymocyte differentiation. LDSWs presumably cause stress forces on the exposed material surface by the high pressure amplitude and the short rise time. LDSWs can be regarded as a kind of mechanical stimulation. What will happen if T cells were exposed to the LDSWs which are able to damage plasma membrane without impairing other organelles? Whether are the LDSWs able to induce the rapid release of ATP from intracellular plasma? And whether is the rapid release of ATP able to elicit the relevant biological effect on T cells?In addition, mechanical stress activates multiple signaling enzymes, including Mitogen activated protein kinase p38 (p38 MAPK). This kinase is structurally related to the yeast protein HOG-1（the products of Saccharomyces cerevisiae osmosensing gene), which is part of the signaling system that allows yeast cells to regulate gene transcription in response to osmotic stress. In human T cells p38 MAPK signaling is involved in t更多还原