本文關(guān)鍵詞：針刀“調(diào)筋治骨”法對KOA兔膝關(guān)節(jié)軟骨FAK-PI3K-AKT通路及股四頭肌力學(xué)性能的影響　出處：《北京中醫(yī)藥大學(xué)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： FAK-PI3K-AKT 股四頭肌 生物力學(xué) 膝骨關(guān)節(jié)炎 針刀

【摘要】：膝骨關(guān)節(jié)炎(Knee Osteoarthritis, KOA)是以疼痛、僵硬、功能障礙為主要臨床表現(xiàn),以軟骨退變?yōu)榈湫筒±砀淖?累及肌肉、肌腱、關(guān)節(jié)囊等膝周軟組織的全膝關(guān)節(jié)退行性疾病,嚴(yán)重時(shí)可導(dǎo)致關(guān)節(jié)畸形,甚至喪失關(guān)節(jié)功能,嚴(yán)重影響患者生活質(zhì)量,耗費(fèi)大量醫(yī)療資源。根據(jù)流行病學(xué)調(diào)查顯示,我國大于65歲的人群中約有60%有X線KOA表現(xiàn),65歲以上人群發(fā)病率高達(dá)85%。關(guān)于KOA的病因尚不明確,也尚無能夠治愈KOA的療法。針刀療法作為一種新興的中醫(yī)微創(chuàng)療法在治療KOA上具有簡、廉、效、安的優(yōu)勢。在已往的研究中,關(guān)于針刀治療KOA的鎮(zhèn)痛、抗炎機(jī)制己得到部分闡釋,且已發(fā)現(xiàn)針刀治療KOA時(shí)獨(dú)特的生物力學(xué)干預(yù)作用以及促進(jìn)軟骨修復(fù)的作用可能是針刀治療KOA療效顯著的主要原因。本研究是依托郭長青教授國家自然科學(xué)基金“針刀“調(diào)筋治骨”法治療膝骨關(guān)節(jié)炎的效應(yīng)機(jī)制及其與FAK-PI3K-AKT力學(xué)信號轉(zhuǎn)導(dǎo)通路的相關(guān)性研究”的部分內(nèi)容,從針刀對軟骨FAK-PI3K-AKT信號通路及股四頭肌生物力學(xué)特性的影響為切入點(diǎn),探討針刀“調(diào)筋治骨”法的療效與機(jī)制。研究目的本實(shí)驗(yàn)以軟骨損傷修復(fù)及其與股四頭肌力學(xué)特性改變之間的關(guān)系為切入點(diǎn),旨在驗(yàn)證針刀促軟骨損傷修復(fù)的分子生物學(xué)機(jī)制及其與針刀影響股四頭肌力學(xué)特性之間的關(guān)系,闡釋針刀通過“調(diào)筋”而“治骨”治療KOA思路的科學(xué)性,為以經(jīng)筋理論為指導(dǎo)下開展針刀治療KOA提供實(shí)驗(yàn)和理論依據(jù)。研究方法在實(shí)驗(yàn)一中,將6月齡28只健康清潔級新西蘭雄兔隨機(jī)分為正常組、模型組、針刀組、電針組,以左后肢伸直位固定法建立KOA兔模型,于造模結(jié)束1周后分別對針刀組和電針組KOA兔進(jìn)行針刀和電針干預(yù)。針刀干預(yù)組選取左后肢股內(nèi)外側(cè)肌、股直肌、股二頭肌肌腱在膝關(guān)節(jié)處的止點(diǎn),鵝足腱囊,條索結(jié)節(jié)處進(jìn)行針刀松解治療,每周2次,治療4周。電針干預(yù)組選取左后肢“梁丘-血�！薄ⅰ皟�(nèi)膝眼-外膝眼”進(jìn)行電針治療,疏密波,頻率2/100Hz,強(qiáng)度3mA,每次20min,每周3次,治療4周。采用LequesneMG膝關(guān)節(jié)級別評估量表、膝關(guān)節(jié)被動活動范圍觀察各組兔行為學(xué)情況，，采用蕃紅-固綠染色在光鏡下觀察各組兔膝關(guān)節(jié)軟骨形態(tài)學(xué)情況并按Mankin法進(jìn)行評分,于掃描電鏡下觀察各組兔膝關(guān)節(jié)軟骨表面超微結(jié)構(gòu)情況,以比較針刀和電針兩種方法干預(yù)KOA兔的療效及差異。在實(shí)驗(yàn)二中,將49只新西蘭雄兔隨機(jī)分為正常組、模型組、模型抑制劑組、針刀組、針刀抑制劑組、電針組、電針抑制劑組,造模與治療方法同實(shí)驗(yàn)一。各抑制劑組均在每次治療前2h采用關(guān)節(jié)腔注射FAK特異性抑制劑PF-562271 (200μM,0.5ml)。采用Western blot和Realtime-PCR檢測軟骨中p-FAK、p-PI3K、p-AKT、COL-Ⅱ、Aggrecan的蛋白和mRNA表達(dá)水平,探討針刀及電針干預(yù)對軟骨合成和分解代謝的影響及其與FAK-PI3K-AKT信號通路的關(guān)系。在實(shí)驗(yàn)三中,分組和干預(yù)方法同實(shí)驗(yàn)一。采用電生理學(xué)方法檢測各組兔左后肢股四頭肌單收縮和強(qiáng)直收縮能力,以SuperSonic Imagine AixPlorer型實(shí)時(shí)SWE超聲診斷儀檢測各組兔左后肢股四頭肌彈性模量,探討針刀及電針干預(yù)對股四頭肌力學(xué)特性的影響。研究結(jié)果1.行為學(xué)結(jié)果：干預(yù)前,與正常組對比,模型組、針刀組、電針組KOA兔左后肢膝關(guān)節(jié)Lequesne MG評分明顯上升(P**0.01),膝關(guān)節(jié)被動活動范圍明顯下降(P**0.01),三組組間對比無差異；干預(yù)后,與模型組對比,針刀組和電針組Lequesne MG評分下降(P##0.01),針刀組膝關(guān)節(jié)被動活動范圍上升(P#0.05)；與電針組對比,針刀組膝關(guān)節(jié)Lequesne MG評分下降(P▲0.05)；膝關(guān)節(jié)被動活動范圍上升(P▲0.05)。2.形態(tài)學(xué)結(jié)果：膝關(guān)節(jié)軟骨蕃紅-固綠染色切片及軟骨掃描電鏡顯示針刀和電針干預(yù)均能不同程度的抑制KOA兔軟骨基質(zhì)的降解,且針刀干預(yù)的效果優(yōu)于電針干預(yù)；Mankin評分比較顯示,與正常組對比,模型組、針刀組、電針組Mankin評分均上升(P棗*0.01)；與模型組對比,針刀組、電針組Mankin評分下降(P##0.01)；與電針組對比,針刀組Mankin評分下降(P▲0.05)。3. Western blot和Real time-PCR檢測結(jié)果：干預(yù)后,與正常組對比,模型組、針刀組、電針組p-FAK和p-PI3K的蛋白和mRNA, p-AKT的蛋白表達(dá)上升(P*0.05或P**0.01)；與模型組比較,針刀組、電針組p-FAK、p-PI3K的蛋白和mRNA, p-AKT的蛋白表達(dá)升高(P#0.05或P##0.01)；與電針組對比,針刀組p-FAK和p-AKT蛋白,p-PI3K的蛋白和mRNA表達(dá)較電針組升高(P▲0.05)；加抑制劑后,p-FAK、p-PI3K的蛋白和mRNA, p-AKT的蛋白表達(dá)降低(P△0.05或P△△0.01)。同時(shí),與正常組對比,模型組、電針組、針刀組Aggrecan、Col-Ⅱ蛋白和mRNA表達(dá)均呈不同程度的下降(P*0.05或P**0.01)；與模型組對比,針刀組和電針組Aggrecan、Col-Ⅱ的蛋白和mRNA表達(dá)升高(P#0.05或P#0.05)；與電針組對比,針刀組Aggrecan、Col-Ⅱ的蛋白和Aggrecan mRNA表達(dá)升高(P▲▲0.01)；加抑制劑后,Aggrecan、Col-Ⅱ的蛋白和mRNA表達(dá)下降(P△0.05或P△△0.01)。4.力學(xué)檢測結(jié)果：干預(yù)后,與正常組對比,模型組、電針組的KOA兔左后肢股四頭肌單收縮能力下降(P***0.01)、強(qiáng)直收縮能力下降(P*0.05)、肌肉彈性模量下降(P*0.05)；與模型組對比,針刀組KOA兔左后肢股四頭肌單收縮能力提升(P##0.01)、強(qiáng)直收縮能力提升(P#0.05)、肌肉彈性模量也上升(P#0.05)；與電針組對比,針刀組KOA兔左后肢股四頭肌肌肉單收縮和強(qiáng)直收縮能力上升(P▲0.05)。研究結(jié)論1.針刀干預(yù)可以改善膝關(guān)節(jié)活動度、減輕疼痛,并減緩軟骨基質(zhì)退變。2.針刀干預(yù)通過激活軟骨細(xì)胞FAK-PI3K-AKT信號通路,促進(jìn)軟骨細(xì)胞的合成代謝。3.針刀干預(yù)能調(diào)節(jié)股四頭肌的收縮能力及彈性模量,部分恢復(fù)股四頭肌的功能。4.針刀干預(yù)能通過調(diào)節(jié)股四頭肌部分力學(xué)性能為膝關(guān)節(jié)軟骨提供良性應(yīng)力刺激,即通過“調(diào)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.
【學(xué)位授予單位】：北京中醫(yī)藥大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R245


本文編號：1346980