针刀“调筋治骨”法对KOA兔膝关节软骨FAK-PI3K-AKT通路及股四头肌力学性能的影响

发布时间：2017-12-28 18:44

  本文关键词：针刀“调筋治骨”法对KOA兔膝关节软骨FAK-PI3K-AKT通路及股四头肌力学性能的影响　出处：《北京中医药大学》2016年博士论文　论文类型：学位论文

  更多相关文章： FAK-PI3K-AKT 股四头肌 生物力学 膝骨关节炎 针刀

【摘要】：膝骨关节炎(Knee Osteoarthritis, KOA)是以疼痛、僵硬、功能障碍为主要临床表现,以软骨退变为典型病理改变,累及肌肉、肌腱、关节囊等膝周软组织的全膝关节退行性疾病,严重时可导致关节畸形,甚至丧失关节功能,严重影响患者生活质量,耗费大量医疗资源。根据流行病学调查显示,我国大于65岁的人群中约有60%有X线KOA表现,65岁以上人群发病率高达85%。关于KOA的病因尚不明确,也尚无能够治愈KOA的疗法。针刀疗法作为一种新兴的中医微创疗法在治疗KOA上具有简、廉、效、安的优势。在已往的研究中,关于针刀治疗KOA的镇痛、抗炎机制己得到部分阐释,且已发现针刀治疗KOA时独特的生物力学干预作用以及促进软骨修复的作用可能是针刀治疗KOA疗效显著的主要原因。本研究是依托郭长青教授国家自然科学基金“针刀“调筋治骨”法治疗膝骨关节炎的效应机制及其与FAK-PI3K-AKT力学信号转导通路的相关性研究”的部分内容,从针刀对软骨FAK-PI3K-AKT信号通路及股四头肌生物力学特性的影响为切入点,探讨针刀“调筋治骨”法的疗效与机制。研究目的本实验以软骨损伤修复及其与股四头肌力学特性改变之间的关系为切入点,旨在验证针刀促软骨损伤修复的分子生物学机制及其与针刀影响股四头肌力学特性之间的关系,阐释针刀通过“调筋”而“治骨”治疗KOA思路的科学性,为以经筋理论为指导下开展针刀治疗KOA提供实验和理论依据。研究方法在实验一中,将6月龄28只健康清洁级新西兰雄兔随机分为正常组、模型组、针刀组、电针组,以左后肢伸直位固定法建立KOA兔模型,于造模结束1周后分别对针刀组和电针组KOA兔进行针刀和电针干预。针刀干预组选取左后肢股内外侧肌、股直肌、股二头肌肌腱在膝关节处的止点,鹅足腱囊,条索结节处进行针刀松解治疗,每周2次,治疗4周。电针干预组选取左后肢“梁丘-血海”、“内膝眼-外膝眼”进行电针治疗,疏密波,频率2/100Hz,强度3mA,每次20min,每周3次,治疗4周。采用LequesneMG膝关节级别评估量表、膝关节被动活动范围观察各组兔行为学情况，，采用蕃红-固绿染色在光镜下观察各组兔膝关节软骨形态学情况并按Mankin法进行评分,于扫描电镜下观察各组兔膝关节软骨表面超微结构情况,以比较针刀和电针两种方法干预KOA兔的疗效及差异。在实验二中,将49只新西兰雄兔随机分为正常组、模型组、模型抑制剂组、针刀组、针刀抑制剂组、电针组、电针抑制剂组,造模与治疗方法同实验一。各抑制剂组均在每次治疗前2h采用关节腔注射FAK特异性抑制剂PF-562271 (200μM,0.5ml)。采用Western blot和Realtime-PCR检测软骨中p-FAK、p-PI3K、p-AKT、COL-Ⅱ、Aggrecan的蛋白和mRNA表达水平,探讨针刀及电针干预对软骨合成和分解代谢的影响及其与FAK-PI3K-AKT信号通路的关系。在实验三中,分组和干预方法同实验一。采用电生理学方法检测各组兔左后肢股四头肌单收缩和强直收缩能力,以SuperSonic Imagine AixPlorer型实时SWE超声诊断仪检测各组兔左后肢股四头肌弹性模量,探讨针刀及电针干预对股四头肌力学特性的影响。研究结果1.行为学结果：干预前,与正常组对比,模型组、针刀组、电针组KOA兔左后肢膝关节Lequesne MG评分明显上升(P**0.01),膝关节被动活动范围明显下降(P**0.01),三组组间对比无差异；干预后,与模型组对比,针刀组和电针组Lequesne MG评分下降(P##0.01),针刀组膝关节被动活动范围上升(P#0.05)；与电针组对比,针刀组膝关节Lequesne MG评分下降(P▲0.05)；膝关节被动活动范围上升(P▲0.05)。2.形态学结果：膝关节软骨蕃红-固绿染色切片及软骨扫描电镜显示针刀和电针干预均能不同程度的抑制KOA兔软骨基质的降解,且针刀干预的效果优于电针干预；Mankin评分比较显示,与正常组对比,模型组、针刀组、电针组Mankin评分均上升(P枣*0.01)；与模型组对比,针刀组、电针组Mankin评分下降(P##0.01)；与电针组对比,针刀组Mankin评分下降(P▲0.05)。3. Western blot和Real time-PCR检测结果：干预后,与正常组对比,模型组、针刀组、电针组p-FAK和p-PI3K的蛋白和mRNA, p-AKT的蛋白表达上升(P*0.05或P**0.01)；与模型组比较,针刀组、电针组p-FAK、p-PI3K的蛋白和mRNA, p-AKT的蛋白表达升高(P#0.05或P##0.01)；与电针组对比,针刀组p-FAK和p-AKT蛋白,p-PI3K的蛋白和mRNA表达较电针组升高(P▲0.05)；加抑制剂后,p-FAK、p-PI3K的蛋白和mRNA, p-AKT的蛋白表达降低(P△0.05或P△△0.01)。同时,与正常组对比,模型组、电针组、针刀组Aggrecan、Col-Ⅱ蛋白和mRNA表达均呈不同程度的下降(P*0.05或P**0.01)；与模型组对比,针刀组和电针组Aggrecan、Col-Ⅱ的蛋白和mRNA表达升高(P#0.05或P#0.05)；与电针组对比,针刀组Aggrecan、Col-Ⅱ的蛋白和Aggrecan mRNA表达升高(P▲▲0.01)；加抑制剂后,Aggrecan、Col-Ⅱ的蛋白和mRNA表达下降(P△0.05或P△△0.01)。4.力学检测结果：干预后,与正常组对比,模型组、电针组的KOA兔左后肢股四头肌单收缩能力下降(P***0.01)、强直收缩能力下降(P*0.05)、肌肉弹性模量下降(P*0.05)；与模型组对比,针刀组KOA兔左后肢股四头肌单收缩能力提升(P##0.01)、强直收缩能力提升(P#0.05)、肌肉弹性模量也上升(P#0.05)；与电针组对比,针刀组KOA兔左后肢股四头肌肌肉单收缩和强直收缩能力上升(P▲0.05)。研究结论1.针刀干预可以改善膝关节活动度、减轻疼痛,并减缓软骨基质退变。2.针刀干预通过激活软骨细胞FAK-PI3K-AKT信号通路,促进软骨细胞的合成代谢。3.针刀干预能调节股四头肌的收缩能力及弹性模量,部分恢复股四头肌的功能。4.针刀干预能通过调节股四头肌部分力学性能为膝关节软骨提供良性应力刺激,即通过“调筋”而“正骨”。
[Abstract]:Knee osteoarthritis (Knee Osteoarthritis KOA) is in pain, stiffness and dysfunction as the main clinical manifestations, with typical pathological changes of cartilage degeneration, muscle involvement, tendon, joint capsule and other soft tissue knee total knee joint degenerative diseases, severe cases can lead to joint deformity and loss of joint function, and serious influence the quality of life of patients, consume a large amount of medical resources. According to the epidemiological survey, about 60% of the people over 65 years old in China have X - ray KOA, and the incidence of the population over 65 years old is up to 85%. The etiology of KOA is not yet clear, and there is no cure for KOA. Acupotomy, as a new kind of traditional Chinese medicine minimally invasive therapy, has the advantages of simple, cheap, effective and safe in the treatment of KOA. In past studies, the analgesic and anti-inflammatory mechanisms of acupotomy for KOA have been partially explained. It has been found that the unique biomechanical intervention and the effect of promoting cartilage repair by needle knife therapy for KOA may be the main reason for the efficacy of acupotomy in the treatment of KOA. Part of the contents of this study is based on the effect mechanism of the natural science foundation of "national professor Guo Changqing" needle knife treatment of bone tendon transfer "in the treatment of knee osteoarthritis research and its correlation with FAK-PI3K-AKT signal transduction pathways, from the effects of acupotomy on cartilage FAK-PI3K-AKT pathway and femoral head four muscle biomechanical characteristics as the breakthrough point, to investigate the effect of" and the mechanism of acupotomy treatment of bone tendon transfer method. Objective to study the relationship between the repair of cartilage injury and femoral head four muscle mechanical properties change as the starting point, to verify the molecular biological mechanism of acupotomy plerosis cartilage injury and the relationship between the femoral head four muscle mechanical properties and effect of needle knife, knife through the interpretation of "scientific tendon transfer" and "to the bone" treatment KOA thought, as the reinforcement theory to carry out under the guidance of acupuncture treatment of KOA to provide experimental and theoretical basis for. Research methods in Experiment 1, the June age 28 healthy clean male New Zealand rabbits were randomly divided into normal group, model group, acupuncture group and acupuncture group, the left hindlimb extended fixation method to establish the KOA rabbit model, rats in 1 weeks after the end of the knife group and EA group KOA rabbits by needle knife and electro acupuncture. In the Acupotomy intervention group, the left hind leg, the medial and lateral femoral muscles, the rectus femoris and the two muscles of the femoris were placed at the ends of the knee joints, and the Acupotomy was used for the treatment of the 2 cases of the treatment of 4 weeks. Electro acupuncture group selected the left hind "Liang Qiu - blood", "in the eye of the knee - knee eye" electro acupuncture treatment, dilatational wave, frequency of 2/100Hz and intensity of 3mA, 20min each time, 3 times a week for 4 weeks. The LequesneMG knee level assessment scale, knee joint passive range of motion was observed in rabbit behavior, using safranin / fast green staining under light microscope to observe rabbit articular cartilage morphology and was assessed by Mankin method in scanning electron microscope observe rabbit articular cartilage surface ultrastructure, intervention effect and the difference of KOA in rabbits to compare two methods of acupotomy and acupuncture. In Experiment 2, 49 New Zealand male rabbits were randomly divided into normal group, model group, model inhibitor group, Acupotomy group, Acupotomy inhibitor group, EA group and EA inhibitor group. The FAK specific inhibitor PF-562271 (200 u M, 0.5ml) was injected into the articular cavity of 2H before each treatment. The expression levels of p-FAK, p-PI3K, p-AKT, COL- II and Aggrecan in cartilage were detected by Western blot and Realtime-PCR. The effects of acupotomy and electroacupuncture on cartilage synthesis and catabolism were discussed, and the relationship between them and FAK-PI3K-AKT signal pathway was also discussed. In the third experiment, the group and the intervention method are the same as the experiment one. Using electrophysiological methods were used to investigate the left femoral head four muscle twitch and tetanic contraction ability, using SuperSonic Imagine AixPlorer real-time SWE ultrasound diagnosis instrument of rabbits left femoral head four muscle elastic modulus, to investigate the effects of acupotomy and acupuncture intervention characteristics of femoral head four strength. Results of the 1. behavioral results: before the intervention, compared with the normal group, model group, acupuncture group and electroacupuncture group KOA rabbits left hind knee joint Lequesne MG score increased significantly (P**0.01), knee joint passive range of motion was significantly decreased (P**0.01), the three groups were compared to no difference; after the intervention, compared with the model group Lequesne, Acupotomy group and electro acupuncture group MG score decreased (P##0.01), Acupotomy group of knee joint passive range of motion (P#0.05); rise compared with the acupuncture group, Acupotomy group Lequesne knee score decreased MG (P ^ 0.05); knee joint passive range of motion (P ^ 0.05) rise. 2. morphological results: knee cartilage safranin fast green staining and scanning electron microscopy showed that cartilage knife and electro acupuncture can inhibit the degradation of KOA rabbit cartilage matrix in different degree. The effect of the electroacupuncture and acupuncture intervention; Mankin score comparison showed that compared with the normal group, model group, acupuncture group and electroacupuncture group Mankin scores were increased (P jujube *0.01); compared with the model group, acupuncture group and electroacupuncture group Mankin score decreased (P##0.01); compared with the electroacupuncture group, Acupotomy group Mankin score decreased (P 0.05). 3. Western blot and Real time-PCR results: after the intervention, compared with the normal group, model group, acupuncture group, EA group p-FAK and p-PI3K protein and mRNA expression of p-AKT protein increased (P*0.05 or P**0.01); compared with the model group, Acupotomy group and electro acupuncture group p-FAK, p-PI3K protein and mRNA expression of p-AKT. The protein level (P#0.05 or P##0.01); compared with the electroacupuncture group, Acupotomy group p-FAK and p-AKT protein, p-PI3K protein and mRNA expression compared with EA group increased (P 0.05); and p-FAK inhibitor, and p-PI3K protein and mRNA p-AKT protein expression decreased (P 0.05 or P delta delta 0.01). At the same time, compared with the normal group, the expression of Aggrecan, Col- II and mRNA in the model group, the electroacupuncture group and the Acupotomy group were in different degrees.
【学位授予单位】：北京中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R245


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