舒筋汤对兔膝关节挛缩模型关节功能和TGF-β1表达的影响
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摘要
目的:研究舒筋汤对兔膝关节挛缩模型的关节功能、关节囊形态以及关节TGF-β1表达的影响。
方法:24只大白兔按Hildebrand法固定右膝关节8周复制创伤后关节挛缩,造模成功后随机分为给药组、模型组和对照组,随机取左侧正常关节作为正常组(n = 8)。给药组、对照组分别予以舒筋汤、正骨水溶液薰洗,模型组不予用药处理,各组均同时予以功能锻炼。给药3、6周末分别测量关节挛缩程度,抽取关节液双夹心抗体Elisa法测量TGF-β1含量。处死动物,观察关节形态,取关节囊,固定后测量关节囊厚度,切片行HE染色进行形态学观察,Masson染色取图进行关节胶原纤维面积定量测定;滑膜组织匀浆,双夹心抗体法Elisa和Real Time-RT PCR测量关节囊滑膜组织TGF-β1表达。检测数据采用SPSS11.0软件进行统计分析。
结果:实验过程中无动物死亡或切口感染,造模后形成关节挛缩。经功能锻炼及给药处理,功能上,各组右膝关节挛缩程度均获显著改善,给药组与对照组、模型组和正常组相比(14.6°:19.6°:22.0°:10.2°),差异显著,除正常组外,其余各组均未恢复正常关节功能。形态学上,模型组和对照组滑膜内壁出现皱缩、增厚、成角及萎缩等改变,给药组介于模型组与正常组之间。关节囊纤维化评价上,给药组关节囊厚度明显低于对照组,高于正常组,差异显著,而与模型组无明显差异,胶原面积方面给药组与各组均存在差异。TGF-β1表达,用药组与对照组、模型组和正常组比较,关节液TGF-β1表达差异在三周明显,而在六周末,差异的显著性降低;关节囊TGF-β1在蛋白质水平上低于对照组和模型组,与正常组无明显差异;mRNA水平上的表达明显低于对照组与模型组,高于正常组,差异显著。
结论:舒筋汤辅助功能锻炼能有效减轻关节囊挛缩、恢复关节功能。在组织学上可减轻挛缩关节关节囊的纤维化及滑膜的退变。减少TGF-β1的表达是舒筋汤抗关节挛缩的机制之一。
Objective To investigate effect of ShuJinTang on joint function, capsule morphous and TGF-β1 expression in rabbit knee joint contracture models.
Methods According to“Hildebrand”method,the right knees of 24 skeletally mature female NEW rabbits were immobilized by 8 weeks to model the post-trauma contracture.The immobilization was discontinued at 8 weeks and the rabbits were divided among three groups: medication administration team(MAT), model team(MT) and control group(CG), the left knees serviced as normal control (NC) (n=8). The knee of MAT and MT were fumagated twice a day by ShuJinTang and ZhengGuShui solution respectively.Functional exercise was applied in all group. Measuring the joint contracture degree and taking suction synovial fluid to measure TGF-β1 by Elisa was conduct at the end of 3 weeks and 6 weeks respectively.And then the capsular membrane was resected.The severity of adhesions was assessed,applying joint capsule thickness. Also the stifle joints were histologically evaluated for fibrosis by HE and Masson dyeing. After tissue bomogenate, TGF-β1 expression of the synovial capsule were evaluated applying double sandwich antibody Elisa and Real TimePCR.
Result No rabbitdied and all K wires were intact after 8 weeks of immobilization. The range of motion measures of the contracture knees showed a loss of extension that averaged 45°before treatment.The function of right knees progressed inequality in every group.The loss of extension showed 14.6°,19.6°,22.0°,10.2°in MAT, CG, MT and NC respectively. There were significant difference when compared MAT with CG, MT and NC.The membrana synovialisof MT show shrinkage,thickening,angulation depaup- -eration in morphologia.The change of MAT show light membrana synovialis degeneration.The green areas and capsule thickness in MAT were small than CG and MT,the discrepancy was significant. TGF-β1 expression in synovial fluid and articular capsule were elevated in right knees than left,and the MAT elevated less than CG and MT. TGF-β1mRNA levels showed similarity change in each group.
Conclusion ShuJinTang incorporation functional exercise can relieve joint contraction,restore joint function. The histological evaluation showed that ShuJinTang could decrease fibration of articular capsule . All the effect may be achived by reduced the expressio of TGF-β1 .
方法:24只大白兔按Hildebrand法固定右膝关节8周复制创伤后关节挛缩,造模成功后随机分为给药组、模型组和对照组,随机取左侧正常关节作为正常组(n = 8)。给药组、对照组分别予以舒筋汤、正骨水溶液薰洗,模型组不予用药处理,各组均同时予以功能锻炼。给药3、6周末分别测量关节挛缩程度,抽取关节液双夹心抗体Elisa法测量TGF-β1含量。处死动物,观察关节形态,取关节囊,固定后测量关节囊厚度,切片行HE染色进行形态学观察,Masson染色取图进行关节胶原纤维面积定量测定;滑膜组织匀浆,双夹心抗体法Elisa和Real Time-RT PCR测量关节囊滑膜组织TGF-β1表达。检测数据采用SPSS11.0软件进行统计分析。
结果:实验过程中无动物死亡或切口感染,造模后形成关节挛缩。经功能锻炼及给药处理,功能上,各组右膝关节挛缩程度均获显著改善,给药组与对照组、模型组和正常组相比(14.6°:19.6°:22.0°:10.2°),差异显著,除正常组外,其余各组均未恢复正常关节功能。形态学上,模型组和对照组滑膜内壁出现皱缩、增厚、成角及萎缩等改变,给药组介于模型组与正常组之间。关节囊纤维化评价上,给药组关节囊厚度明显低于对照组,高于正常组,差异显著,而与模型组无明显差异,胶原面积方面给药组与各组均存在差异。TGF-β1表达,用药组与对照组、模型组和正常组比较,关节液TGF-β1表达差异在三周明显,而在六周末,差异的显著性降低;关节囊TGF-β1在蛋白质水平上低于对照组和模型组,与正常组无明显差异;mRNA水平上的表达明显低于对照组与模型组,高于正常组,差异显著。
结论:舒筋汤辅助功能锻炼能有效减轻关节囊挛缩、恢复关节功能。在组织学上可减轻挛缩关节关节囊的纤维化及滑膜的退变。减少TGF-β1的表达是舒筋汤抗关节挛缩的机制之一。
Objective To investigate effect of ShuJinTang on joint function, capsule morphous and TGF-β1 expression in rabbit knee joint contracture models.
Methods According to“Hildebrand”method,the right knees of 24 skeletally mature female NEW rabbits were immobilized by 8 weeks to model the post-trauma contracture.The immobilization was discontinued at 8 weeks and the rabbits were divided among three groups: medication administration team(MAT), model team(MT) and control group(CG), the left knees serviced as normal control (NC) (n=8). The knee of MAT and MT were fumagated twice a day by ShuJinTang and ZhengGuShui solution respectively.Functional exercise was applied in all group. Measuring the joint contracture degree and taking suction synovial fluid to measure TGF-β1 by Elisa was conduct at the end of 3 weeks and 6 weeks respectively.And then the capsular membrane was resected.The severity of adhesions was assessed,applying joint capsule thickness. Also the stifle joints were histologically evaluated for fibrosis by HE and Masson dyeing. After tissue bomogenate, TGF-β1 expression of the synovial capsule were evaluated applying double sandwich antibody Elisa and Real TimePCR.
Result No rabbitdied and all K wires were intact after 8 weeks of immobilization. The range of motion measures of the contracture knees showed a loss of extension that averaged 45°before treatment.The function of right knees progressed inequality in every group.The loss of extension showed 14.6°,19.6°,22.0°,10.2°in MAT, CG, MT and NC respectively. There were significant difference when compared MAT with CG, MT and NC.The membrana synovialisof MT show shrinkage,thickening,angulation depaup- -eration in morphologia.The change of MAT show light membrana synovialis degeneration.The green areas and capsule thickness in MAT were small than CG and MT,the discrepancy was significant. TGF-β1 expression in synovial fluid and articular capsule were elevated in right knees than left,and the MAT elevated less than CG and MT. TGF-β1mRNA levels showed similarity change in each group.
Conclusion ShuJinTang incorporation functional exercise can relieve joint contraction,restore joint function. The histological evaluation showed that ShuJinTang could decrease fibration of articular capsule . All the effect may be achived by reduced the expressio of TGF-β1 .
引文
1.Hildebrand KA, Sutherland C, Zhang M. Rabbit knee model of post-traumatic joint contractures: the long-term natural history of motion loss and myo?broblasts. J Orthop Res,2004. 22:313–320
2.Livak KJ, Schmittgen TD.Analysisof relative gene expression data using realtime quantitative PCR and the 2 ( - Delta Delta C (T) )Method Methods,2001,25(4): 402 - 408.
3.苗明山。实验动物和动物实验技术[M]。北京:中国中医药出版社,1997;260
4.俞力行,张友定,姜宏.熏洗方对大鼠骨折后期僵硬关节滑膜型胶原mRNA及TGF-β表达的影响.中医正骨, 2005, 17( 1) : 7 9
5.Kevin A. Hildebrand,Mei Zhang,Niccole M. Germscheid.Cellular, matrix, and growth factor components of the joint capsule are modified early in the process of posttraumatic contracture formation in a rabbit model.Acta Orthopaedica ,2008; 79 (1): 116–125
6.Trudel G, Uhthoff HK.Contractures secondary to immobility: is the restriction articular or muscular? An experimental longitudinal study in the rat knee. Arch Phys Med Rehabil,2000,81:6–13
7 Saraiya H. Management of an unusual extreme extension contracture of the wrist: role of a custom-designed exercise program in achieving a good range of movement and prevention of recontraction. J Burn Care Rehabil. 2003 ,24(6):378-381.
8.Hildebrand KA, Zhang M, Hart DA. High rate of joint capsule matrix turnover in chronic human elbow contractures. Clin Orthop Relat Res.2005 Oct;439:228-234.
9何承建,曾俊华,魏玉玲,等。当归预防椎板切除术后硬膜外粘连的实验研究。中国中医骨伤科杂志,2007,15 (7 ):53-55
10韩剑宇,都立君,庞建华,红花对增生性癖痕成纤维细胞增殖和胶原合成的影响。哈尔滨医科大学学报,2005,39(4):337-339
11.Okazaki R, Sakai A, Uezono Y, et al . Sequential changes in transforming growth factor (TGF)-β1 concentration in synovial fluid and mRNA expression of TGFβ1 receptors in chondrocytes after immobilization of rabbit knees.J BoneMiner Metab,2001,19(4):228-235
12.Allen JB, Manthey CL, Hand AR, et al . Rapid onset of synovial inflammation and hyperplasia induced by transforming growth factor beta..J Exp Med, 1990,171(1):231-247.
13.Hildebrand KA,Mei Zhang, Niccole M. et al. Cellular, matrix, and growth factor components of the joint capsule are modified early in the process of posttraumatic contracture formation in a rabbit model。Acta Orthopaedica,2008,79(1):116-125
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2.Livak KJ, Schmittgen TD.Analysisof relative gene expression data using realtime quantitative PCR and the 2 ( - Delta Delta C (T) )Method Methods,2001,25(4): 402 - 408.
3.苗明山。实验动物和动物实验技术[M]。北京:中国中医药出版社,1997;260
4.俞力行,张友定,姜宏.熏洗方对大鼠骨折后期僵硬关节滑膜型胶原mRNA及TGF-β表达的影响.中医正骨, 2005, 17( 1) : 7 9
5.Kevin A. Hildebrand,Mei Zhang,Niccole M. Germscheid.Cellular, matrix, and growth factor components of the joint capsule are modified early in the process of posttraumatic contracture formation in a rabbit model.Acta Orthopaedica ,2008; 79 (1): 116–125
6.Trudel G, Uhthoff HK.Contractures secondary to immobility: is the restriction articular or muscular? An experimental longitudinal study in the rat knee. Arch Phys Med Rehabil,2000,81:6–13
7 Saraiya H. Management of an unusual extreme extension contracture of the wrist: role of a custom-designed exercise program in achieving a good range of movement and prevention of recontraction. J Burn Care Rehabil. 2003 ,24(6):378-381.
8.Hildebrand KA, Zhang M, Hart DA. High rate of joint capsule matrix turnover in chronic human elbow contractures. Clin Orthop Relat Res.2005 Oct;439:228-234.
9何承建,曾俊华,魏玉玲,等。当归预防椎板切除术后硬膜外粘连的实验研究。中国中医骨伤科杂志,2007,15 (7 ):53-55
10韩剑宇,都立君,庞建华,红花对增生性癖痕成纤维细胞增殖和胶原合成的影响。哈尔滨医科大学学报,2005,39(4):337-339
11.Okazaki R, Sakai A, Uezono Y, et al . Sequential changes in transforming growth factor (TGF)-β1 concentration in synovial fluid and mRNA expression of TGFβ1 receptors in chondrocytes after immobilization of rabbit knees.J BoneMiner Metab,2001,19(4):228-235
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