摘要
目的采用“电击跳跃法”建立大鼠跟腱末端病模型,并对模型进行火针治疗。观察末端区组织和细胞形态变化、HSP70表达和细胞凋亡指数变化,依据末端结构特点从病因、病理角度探讨火针治疗末端病的作用机理,为末端病的火针疗法提供理论依据。方法以1月龄雄性SD大鼠为研究对象,通过“电击跳跃法”建立跟腱末端病模型。将88只大鼠随机分为3大组,分别为：正常组,治疗组(治疗1、2、3、4、5组)和对照组(对照1、2、3、4、5组)。造模后进行火针治疗,于1次治疗后第1、2、3、4天分别处死治疗1、2、3、4组及其相应对照组,再于4次治疗后第4天处死治疗5组和对照5组,取每只大鼠单侧后足跟腱和完整的跟腱末端区组织,经固定、脱钙、包埋、切片、贴片、烤片后,常规HE染色,观察末端区组织和细胞形态变化；采用免疫组化ABC法检测末端区跟腱、纤维软骨区、钙化软骨区、跟骨、腱骨关节面软骨的HSP70阳性率；应用TUNEL法检测跟腱、纤维软骨区、钙化软骨区、跟骨、腱骨关节面软骨、腱围的细胞凋亡指数(AI)。所有数据用SPSS13.0进行统计分析。
结果(1)HE染色镜下观察：经1次火针治疗后,治疗1、2、3组大鼠跟腱末端区与对照组相比,在病理上无明显变化。治疗4组在腱围、跟腱等部位的病理有所改善,但差异并不显著。而经4次火针治疗后,治疗5组从病理上看明显减轻,更接近于正常组。(2)HSP70表达：1次火针治疗后,治疗组大鼠末端区主要在纤维软骨区、腱骨关节面软骨两个部位的HSP70阳性率比对照组有不同程度升高；经4次火针治疗后,纤维软骨区、腱骨关节面软骨两个部位的HSP70阳性率明显高于对照组；4次火针治疗后在纤维软骨区、腱骨关节面软骨两个部位的HSP70阳性率明显高于1次火针治疗。(3)细胞凋亡检测：1次火针治疗后,治疗组大鼠末端区主要在跟腱、纤维软骨区、腱围这三个部位的AI比对照组有不同程度降低；经4次火针治疗后,跟腱、纤维软骨区、腱骨关节面软骨、腱围的AI明显低于对照组；4次火针治疗后在跟腱、纤维软骨区、腱骨关节面软骨、腱围的AI明显低于1次火针治疗。
结论(1)8周“电击跳跃法”造模训练成功地建立了大鼠跟腱末端病模型。(2)1次火针治疗诱导末端区合成大量HSP70,主要表现在纤维软骨区、腱骨关节面软骨两个部位,且在针刺后当天即达到一个高峰,之后3天呈连续下降趋势。4次火针治疗后这两个部位的HSP70合成量大大增加,HSP70阳性率明显升高,4次火针治疗能更进一步促进末端病的恢复。(3)1次火针治疗加速了跟腱、纤维软骨区、腱围三个部位凋亡细胞的吸收和清除,促进了末端区组织和细胞的修复。而4次火针治疗后,在跟腱、腱骨关节面软骨、纤维软骨区、腱围等多个部位的细胞凋亡指数都发生了改变,末端区的整体结构得到了恢复,4次火针治疗在促进末端病的整体恢复上效果更好。
Purpose Establish the rat model of Achilles Tendon's enthesiopathy by "electric shock jump".Then treat the model rats by using heated needling.By observing the terminal region of cell and tissue morphology, expression of HSP70 and change of cell apoptosis index, discuss the mechanisiam of heated needling therapy on enthesiopathy from a pathological point of view so as to provide a theoretical basis for heated needling therapy.
Methods Take 1-month-old male SD rats as the object for study. By "electric shock jump" establish the rat model of Achilles Tendon's enthesiopathy. The 88 rats were randomly divided into 3 groups, namely:the normal group, treatment group (treatment group 1,2,3,4,5) and control group (control group 1,2,3,4,5).After modeling, treat the rats by heated needling.Then kill each group of rats and the corresponding control group at the 1st,2nd,3rd,4th day after the first treatment and the last group and its control group at the fourth day after four times treatment. Cut unilateral hind tendon terminal region of the Achilles tendon and complete organization,then fixed,decalcified,embedded,cut,patch and baked it. After conventional HE staining,observe the terminal region of cell and tissue morphology. Using immunohistochemistry of ABC to detect positive rate of HSP70 at T,UF,MF,C and CA. Apply TUNEL to detect the apoptosis index(AI)of T,UF,MF, C,CA and PT. All datas are analyzed with SPSS13.0.
Results (1) HE staining microscopic observation:After one time of heated needling treatment,compared with the corresponding control group,terminal region of Achilles tendon of treat group 1,2,3 rats has no significant change in the pathology. The treat group 4 has largely improved at T,CA and other parts of the Achilles tendon in the pathology, but the difference was not significant. After treated by heated needling therapy for 4 times, treat group 5 significantly reduced from the pathological point of view, closer to the normal group. (2) The expression of HSP70:After 1 time heated needling therapy, HSP70-positive rate of the treatment group in the terminal region mainly at the parts of UF and CA increased in varying degrees than the control group;(2) After 4 times heated needling therapy, HSP70-positive rate of the treatment group at the parts of UF and CA was significantly higher than the control group. HSP70-positive rate of the treatment group at the parts of UF and CA after 4 times heated needling therapy was significantly higher than the 1 time heated needling therapy groups. (3) The detection of apoptosis:After 1 time heated needling therapy, AI of the treatment group in the terminal region mainly at the parts of T,UFand PT decreased in varying degrees than the control group; After 4 times heated needling therapy, AI of the treatment group at the parts of T,UF, CA and PT was significantly lower than the control group; AI of the treatment group at the parts of T,UF, CA and PT after 4 times heated needling therapy was significantly lower than the 1 time heated needling therapy groups.
Conclusions (1) Training by "electric shock jump" for 8 weeks successfully established the rat model of Achilles Tendon's enthesiopathy. (2)1 time heated needling therapy can cause the tissues and cells of tenninal region to induce a large number of synthesis of HSP70 mainly at the two parts of UF and CA, and reached a peak on the therapy day, then showed a continuous downward trend during 3 days after therapy.After 4 times heated needling therapy, the synthesis of HSP70 on the two parts largely increased and positive rate of HSP70 also increased,further enhanced the recovery of enthesiopathy. (3) 1 time heated needling therapy accelerated the purge and absorption of apoptosis cells at T,UF and PT, promoted some tissues and cells repairing. After 4 times heated needling therapy,the main parts of T,CA,UF and PT had taken place great changes.So it is better for the overall restoration.
引文
[1]秦岭,胡声宇,陈启明.体育生物医学基础研究与进展[M].北京：人民体育出版社,2001：162-173
[2]曲绵域,于长隆.实用运动医学[M].第4版.北京：北京大学医学出版社,2003：500-502
[3]Becker W, et al.Die Tendopathien, Thieme, Stuttgart,1978,1-96
[4]于长隆.末端病[J].中国运动医学杂志,1984,3(3)：180-185
[5]吕丹,金玉兰,陈刚.股四头肌和小腿三头肌及其肌止装置包括骸骨与跟骨区域慢性运动损伤的模拟实验研究[J].中国运动医学杂志,1982,1(1)：14-19
[6]陈世益,James H C, Wang Savio L Y. Woo.腱病的基础与临床若干研究[J].国外医学·骨科学分册,2005,26(2)：83-86
[7]张世明,罗永宁,刘振寰,等.人体髌骨腱末端结构的组织形态学研究[J].中国运动医学杂志,1985,4(4)：214-218
[8]扈盛,胡亚哲.末端病潮线涨潮的力学分析[J].中国运动医学杂志,2002,21(4)：346-348
[9]于长隆,曲绵域,田小明.免跟腱末端的实验病理研究[J].中国运动医学杂志,1983,2(3)：8-12
[10]黄昌林,张建党等.训练模式对跟腱组织结构的影响及其意义[J].人民军医,2004,47(9)：499-501
[11]黄昌林,钟汉馨,张建党.不同训练模式对跟腱末端纤维软骨细胞影响的实验研究[J].实用医药杂志,2004,3(21)：241-242
[12]扈盛,胡亚哲.热休克蛋白70在末端病中表达的研究[J].中国运动医学杂志,2002,21(3)：232-234
[13]胡亚哲,扈盛,王梅,等.大鼠跟腱末端病的细胞凋亡的检测[J].中国中医骨伤科杂志,2000,8(1)：9-11
[14]扈盛,滕宇,胡亚哲,等.内皮型一氧化氮合酶在大鼠末端病中的表达[J].中国运动医学杂志,2005,24(6)：690-691
[15]王和平,滕宇,胡亚哲,等.诱导型一氧化氮合酶在末端病大鼠跟腱末端区表达及分布的规律[J].中国临床康复医学,2005,9(24)：160
[16]Heinz-R. Kohler, Basim Rahman, Sybille Graff, et al. Expression of the stress-70 protein family due to heavy metal contamination in the slug, deroceras reticulatum, an approach to monitor sublethal stress condition[J]. Chemosphere,1996,33(7):1327-1340
[17]Schlesinger MJ. Heart Shock Proteins:The Search for Functions[J].J Cell Biol,1986.103(2):321-325
[18]Morimoto RI.Tissieres A. Cesrgopoulos C. Stress Proteins in Biology and Medicine[M].Cold Spring Harbor:CSHL Prss.1990:1-36
[19]Sato s, Abe K. Kawgoe J, et al. Isolation of complementary DNAs for heat shock protein(HSP)70 and heat shock cognate protein (HSC)70 genes and the expressions in post-ischaemic gerbil brain. Neurol Res.1992,14:375-380
[20]李冬严,王蕴红.热休克蛋白70及其在运动医学领域的研究进展[J].首都体育学院学报,2002,14(3)：83-86
[21]Hightower LE. Cultured animal cells exposed to amino acid analogues or puromycin rapidly synthesis severa polypeptide[J].J cell Phvsiol.1980. 102(3):407
[22]金惠铭,主编.病理生理学[M].第五版.北京：人民卫生出版社,2002：178-190
[23]Lennon SV, Martin SJ, Cotter TG. Dose-dependent induction of apoptosis in tumor cell lines by widely divergent stimuli[J].Cell Prolif,1991, 24:203-214
[24]胡野,凌志强,主编.细胞凋亡的分子医学[M].北京：军事医学科学出版社,2002
[25]周未艾.细胞凋亡研究进展与在运动医学中应用展望[J].体育科学,1998,18(6)：69-72
[26]曹炜,牛建昭.细胞凋亡研究概况[J].解剖学报,1995,26(4)：443
[27]程欣,沈伟哉.细胞凋亡研究进展[J].解剖学报,1998,21(4)：368
[28]陈东风,郑小飞,等.军事训练所致髌尖末端病的流行病学调查[J].中国现代医学杂志,2003,13(12)：54-55
[29]黄昌林,张建党,薛刚.不同训练模式对跟腱组织结构影响的实验研究及其对军事训练的指导意义[J].解放军医学杂志,2004,4(29)：292-294
[30]Olivieri I, Barozzi L, Padula A. Enthesiopathy:clinical manifestations, imaging and treatment[J]. Bailliere's Clinical Rheumatology,1998 12(4):665-81
[31]任凯,龚晓明.运动员腱止点末端病的回顾与展望[J].中国康复医学杂志,2006,2(18)：754-756
[32]李凤素.以阿是穴为主,采用平刺与围刺法治疗跟腱慢性损伤126例临床分析[J].中国运动医学杂志,2005,24(2)：218-220
[33]刘波.电针治疗腱末端病的生物力学研究-Ⅰ电针对末端结构断裂特性的影响[J].中国中医骨伤科杂志,1995,5(3)：9-13
[34]刘波.电针治疗腱末端病的生物力学研究-Ⅱ电针对末端结构粘弹性的影响[J].中国中医骨伤科杂志,1995,6(3)：4-8
[35]井上基浩.电针刺激对大鼠跟腱血液的影响[J].国外医学中医中药分册,2004,26(6)：359-360
[36]郑良孝,管春燕,李瑛.火针疗法治疗肱骨外上髁炎92例[J].上海针灸杂志,2006,25(5)：7
[37]史清钊.推拿与火针治疗肱骨外上髁炎的疗效比较[J].山东中医药大学学报,2006,30(3)：217
[38]李共信,张锡三,陈淑彦,等.火针治疗肩关节周围炎临床观察[J].上海针灸杂志.2008,27(9)：27-28
[39]高秀领,张雪娟,王俊玲.火针治疗肩关节周围炎28例临床观察[J].河北中医,2008,30(7)：739-940
[40]韦英才.火针配合拔火罐治疗肩周炎疗效观察[J].中国民族医药杂志,2008,9(9)：16-17
[41]宫育卓,林剑鸣,王晓玲.火针治疗跟痛症39例疗效观察[J].新中医,2008,40(4)：78
[42]张晓霞,吴之煌,董明霞.火针疗法治病机理初探[J].北京中医2007,26(9)：576-578.
[43]吴峻,沈蓉蓉.火针治疗慢性软组织损伤的实验研究[J].中国针灸,2002,22(1)：31
[44]吴峻,喻海忠,沈蓉蓉,等.火针治疗慢性软组织损伤实验研究生化检测报告[J].江苏中医药,2003,24(4)：83
[45]严愉芬.近十年火针治疗炎症性疾病的研究概况[J].中国中医药科技,2006,13(4)：283-286
[46]马建.我国末端病研究的历史与现状[J].中医正骨,1992,4(4)：32-33
[47]晋松,冯媛,胡幼平.末端病非手术治疗的中西医研究概况[J].四川中医,2006,24(12)：39-41
[48]吕红斌,王嘉芙.基因治疗在运动医学中的应用[J].中国运动医学杂志,1999,18(3)：254-255
[49]李宏伟.末端病的基因治疗初探[J].浙江体育科学,2003,25(1)：19-21
[50]Schneider W, Niehus W, Knahr K. Hagluhd's syndrome:disappointing results following surgery:a clinical and radiographic analysis [J].Foot Ankle Int, 2000,2:26-30
[51]Marilyn L, Yodlowski, M D, et al. Surgical treatment of achilles tendinitis by decompression of the retrocalcaneal bursa and the superior calcaneal tuberosity [J].Am J Sports Med,2002,30(3):318-321
[52]扈盛,欧高志,滕宇,等.末端病造模方法的研究[J].武汉体育学院学报,2006,40(4)：79-81
[53]姚小卫,扈盛,欧高志,等.末端病大鼠模型的建立[J].中国中医骨伤科杂志,2006,14(5)：17-18
[54]Welch W J, suhan J P. Cellular and biochemical events inmammalian cells during and after recovery from physiological stress[J].J Cell Biol,1986, 103(5):2035-2052
[55]Jacquier—Sarlin M R, Fuller K, Dinh—Xuan A T et al. Experientia[J], 1994,50:1031-1038
[56]扈盛,胡亚哲.运动前热水浴预防末端病的实验研究[J].中国中医骨伤科杂志,2006,11(14)：88-90
[57]郭军,陈杰林.热休克预处理预防跟腱末端病实验性研究[J].医学研究杂志,2006,35(8)：70-71