發(fā)布時(shí)間：2018-03-30 15:10

  本文選題：肌腱微損傷　切入點(diǎn)：牽伸載荷　出處：《第三軍醫(yī)大學(xué)》2017年碩士論文

【摘要】：研究背景:肌腱微損傷是運(yùn)動(dòng)員和普通勞動(dòng)者中廣泛存在的組織損傷,常發(fā)生于反復(fù)、過度拉伸肌腱之后,表現(xiàn)為肌腱局部疼痛、腫脹和運(yùn)動(dòng)功能受限,組織學(xué)表現(xiàn)為肌腱纖維紊亂、斷裂。肌腱微損傷不能有效修復(fù)被認(rèn)為是誘發(fā)肌腱退變、肌腱病的主要原因。有報(bào)道顯示肩袖腱病在普通人中發(fā)病率為2.4~20%,跟腱病在高水平運(yùn)動(dòng)員中發(fā)病率高達(dá)52%,這對(duì)人們的日常生活和運(yùn)動(dòng)員的競(jìng)技水平產(chǎn)生了嚴(yán)重的影響。因此,研究微損傷修復(fù)的影響因素,尋找促進(jìn)微損傷修復(fù)方法是目前仍有待解決的問題。肌腱作為肌肉與骨之間的力學(xué)傳導(dǎo)組織,牽伸載荷對(duì)肌腱生理功能的維持起到重要作用。有學(xué)者認(rèn)為,長期過度牽拉肌腱,使肌腱的損傷無法有效修復(fù)是導(dǎo)致肌腱病的重要原因。同時(shí)也有研究發(fā)現(xiàn),通過離心性牽伸訓(xùn)練可以緩解甚至治愈肌腱病。由此可見,牽伸載荷如一柄雙刃劍,即可導(dǎo)致肌腱損傷,又能促進(jìn)肌腱修復(fù)。所以,研究牽伸載荷對(duì)肌腱的作用機(jī)制,發(fā)揮其有利作用,避免其不利影響,是促進(jìn)肌腱微損傷修復(fù)的可靠途徑。目的:1.探討不同牽伸載荷條件對(duì)大鼠微損傷跟腱修復(fù)的影響,明確有利于肌腱修復(fù)的應(yīng)力強(qiáng)度,闡明理想牽伸載荷對(duì)TSCs生物學(xué)特性的影響。2.闡明牽伸載荷早期對(duì)TSCs立早基因表達(dá)的影響。方法:1.大鼠跟腱微損傷動(dòng)物模型的建立將6只8周齡雄性SD大鼠麻醉后,右側(cè)跟腱注射膠原酶溶液作為實(shí)驗(yàn)組,左側(cè)跟腱注射PBS溶液作為對(duì)照組。注射1周后通過跟腱組織的大體表現(xiàn)、組織病理學(xué)表現(xiàn)和分化標(biāo)志基因表達(dá)對(duì)比,判斷造模是否成功。2.牽伸載荷對(duì)大鼠跟腱微損傷修復(fù)的影響將72只SD大鼠經(jīng)過適應(yīng)性跑臺(tái)訓(xùn)練后,跟腱注射I型膠原酶溶液,制備跟腱微損傷大鼠模型。然后依據(jù)跑臺(tái)強(qiáng)度條件將大鼠隨機(jī)分為3組:對(duì)照組(籠中飼養(yǎng))、低強(qiáng)度組(13m/min,20min/day)、高強(qiáng)度組(17m/min,1h/day)。在跑臺(tái)開始時(shí)、跑臺(tái)1周、跑臺(tái)4周時(shí)分別觀察大鼠跟腱的大體變化、組織病理學(xué)變化、生物力學(xué)變化以及成肌腱分化標(biāo)志基因表達(dá)變化。觀察不同跑臺(tái)條件的牽伸載荷強(qiáng)度對(duì)微損傷跟腱修復(fù)的影響,明確有利于跟腱修復(fù)的理想牽伸載荷強(qiáng)度和時(shí)間點(diǎn)。然后利用該理想牽伸載荷強(qiáng)度和時(shí)間點(diǎn),對(duì)9只8周齡SD雄性大鼠進(jìn)行實(shí)驗(yàn)干預(yù),并分別將提取的TSCs標(biāo)記位為:對(duì)照組(跟腱注射PBS溶液)、誘導(dǎo)組(跟腱注射I型膠原酶溶液)、誘導(dǎo)+跑臺(tái)組(跟腱注射I型膠原酶溶液+理想強(qiáng)度跑臺(tái))。觀察干預(yù)對(duì)TSCs的生物學(xué)影響:結(jié)晶紫染色觀察集落數(shù)量;倒置相差顯微鏡觀察集落形態(tài);CCK8法觀察增殖能力;流式細(xì)胞學(xué)觀察細(xì)胞凋亡和細(xì)胞表面標(biāo)志物;免疫熒光觀察干性標(biāo)志物;細(xì)胞學(xué)染色、RT-PCR觀察細(xì)胞分化能力變化。3.體外牽伸載荷對(duì)大鼠跟腱來源TSCs立早基因表達(dá)的影響沿用本課題組成熟的TSCs分離、培養(yǎng)、傳代技術(shù),利用自制細(xì)胞單軸循環(huán)牽拉裝置在體外對(duì)細(xì)胞進(jìn)行牽拉。分別用2%~12%的牽拉強(qiáng)度和不同牽拉時(shí)間對(duì)細(xì)胞進(jìn)行分組,利用RT-PCR觀察細(xì)胞在牽拉早期c-fos基因、分化標(biāo)志基因表達(dá)的特點(diǎn)。結(jié)果:1.I型膠原酶溶液誘導(dǎo)的大鼠跟腱組織與對(duì)照組相比,大體觀察表現(xiàn)為腱旁組織增多,肌腱黯淡缺乏光澤。組織學(xué)表現(xiàn)為異形細(xì)胞增多、膠原纖維排列紊亂、纖維損傷等。組織成肌腱標(biāo)志基因(Col I、TNMD)表達(dá)明顯減少(p0.05),成骨分化標(biāo)志基因(Runx2)表達(dá)增多(p0.05)。2.對(duì)肌腱微損傷模型大鼠經(jīng)過1周的不同強(qiáng)度跑臺(tái)負(fù)荷刺激后,各實(shí)驗(yàn)組大鼠的跟腱在大體觀察、組織病理學(xué)表現(xiàn)及生物力學(xué)表現(xiàn)等方面未觀察到明顯變化(p0.05),但低強(qiáng)度組跟腱的成肌腱分化標(biāo)志基因(Col I、TNMD)相對(duì)表達(dá)量較對(duì)照組和高強(qiáng)度組均升高(p0.05)。經(jīng)過4周跑臺(tái)牽伸載荷刺激后,各實(shí)驗(yàn)組間出現(xiàn)明顯差異:大體觀察,低強(qiáng)度組腱旁增生組織較對(duì)照組和高強(qiáng)度組明顯減少;組織學(xué)觀察,低強(qiáng)度組組織學(xué)半定量評(píng)分顯著低于對(duì)照組和高強(qiáng)度組,而高強(qiáng)度組的組織學(xué)評(píng)分明顯高于對(duì)照組和低強(qiáng)度組(p0.05);生物力學(xué)測(cè)試,低強(qiáng)度組與對(duì)照組相比,最終應(yīng)力與抗拉強(qiáng)度均明顯升高(p0.05);低強(qiáng)度組的組織成肌腱標(biāo)志基因Col I較對(duì)照組和高強(qiáng)度組明顯升高(p0.05),各組間TNMD基因相對(duì)表達(dá)量未見明顯差異(p0.05)。3.誘導(dǎo)+跑臺(tái)組的TSCs細(xì)胞集落數(shù)較誘導(dǎo)組減少,集落中的細(xì)胞分部較誘導(dǎo)組更緊湊,CCK-8法檢測(cè)到24 h和48 h的誘導(dǎo)+跑臺(tái)組TSCs的OD值較誘導(dǎo)組增高(p0.05)。細(xì)胞凋亡實(shí)驗(yàn)在各實(shí)驗(yàn)組間無明顯差異。誘導(dǎo)+跑臺(tái)組TSCs的細(xì)胞干性標(biāo)記物Nanog較誘導(dǎo)組表達(dá)增多(p0.05)。誘導(dǎo)+跑臺(tái)組TSCs的細(xì)胞標(biāo)志物中CD44、CD73比例較對(duì)照組和誘導(dǎo)組升高。誘導(dǎo)+跑臺(tái)組TSCs的成骨分化標(biāo)志基因(Runx2、Dlx5)、成軟骨分化標(biāo)志基因(Col II)相對(duì)表達(dá)量較誘導(dǎo)組明顯減少(p0.05);而誘導(dǎo)+跑臺(tái)組成肌腱分化標(biāo)志基因(Col I、TNMD)、成脂分化標(biāo)志基因(ap2、PPARγ)相對(duì)表達(dá)量較誘導(dǎo)組明顯增多(p0.05)。經(jīng)過成骨誘導(dǎo)培養(yǎng)后,誘導(dǎo)+跑臺(tái)組的TSCs成骨分化標(biāo)志基因(Runx2、Dlx5)相對(duì)表達(dá)量較對(duì)照組和誘導(dǎo)組顯著升高(p0.05),誘導(dǎo)組的Runx2相對(duì)表達(dá)量較對(duì)照組明顯降低(p0.05);經(jīng)過成脂肪誘導(dǎo)培養(yǎng)后,誘導(dǎo)+跑臺(tái)組TSCs的成脂肪分化標(biāo)志基因(ap2、PPARγ)相對(duì)表達(dá)量較誘導(dǎo)組明顯升高(p0.05),其中PPARγ相對(duì)表達(dá)量較對(duì)照組亦顯著升高(p0.05),誘導(dǎo)組TSCs的PPARγ相對(duì)表達(dá)量較對(duì)照組顯著降低(p0.05);經(jīng)過成軟骨誘導(dǎo)培養(yǎng)后,誘導(dǎo)+跑臺(tái)組TSCs成軟骨分化標(biāo)志基因(Col II、Sox9)的相對(duì)表達(dá)量較誘導(dǎo)組明顯降低(p0.05);而誘導(dǎo)組TSCs的Col II、Sox9基因相對(duì)表達(dá)量較對(duì)照組、誘導(dǎo)+跑臺(tái)組均明顯上升(p0.05)4.與對(duì)照組相比,TSCs在4%和8%牽拉強(qiáng)度下在單軸循環(huán)牽拉30 min時(shí)c-fos mRNA相對(duì)表達(dá)量達(dá)到峰值(p0.05)。2%牽拉強(qiáng)度即可使TSCs的c-fos m RNA相對(duì)表達(dá)量升高(p0.05),6%、8%、12%牽拉強(qiáng)度可使其相對(duì)表達(dá)量進(jìn)一步升高(p0.05)。時(shí)間方面,僅牽拉5 min即可使c-fos相對(duì)表達(dá)量明顯升高(p0.05)。8%牽拉強(qiáng)度在120 min時(shí),Col I、TNMD、Dlx5、Runx2等基因相對(duì)表達(dá)量均升高(p0.05)。結(jié)論:1.I型膠原酶溶液注射入SD大鼠跟腱后能夠在大體和組織病理學(xué)表現(xiàn)上模擬肌腱微損傷表現(xiàn),該模型是一種比較理想的動(dòng)物模型。2.不同強(qiáng)度牽伸載荷在1周對(duì)微損傷修復(fù)的影響無差異。低強(qiáng)度(13m/min,20min/day)跑臺(tái)牽伸載荷4周能夠促進(jìn)大鼠微損傷跟腱的修復(fù);高強(qiáng)度(17m/min,1h/day)跑臺(tái)牽伸載荷4周阻礙微損傷肌腱的修復(fù)。因此對(duì)大鼠微損傷跟腱的修復(fù)來說,13m/min,20min/day跑臺(tái)4周是較為理想的牽伸載荷條件。3.理想牽伸載荷條件對(duì)大鼠跟腱的TSCs影響主要表現(xiàn)在增殖能力強(qiáng)、集落形態(tài)集中、細(xì)胞干性增強(qiáng)、CD44和CD73表達(dá)增多、細(xì)胞向成肌腱和成脂肪方向分化等方面,說明牽伸載荷對(duì)TSCs增殖和分化具有明顯的調(diào)控作用。4.體外單軸循環(huán)牽拉TSCs實(shí)驗(yàn)中,2%牽拉強(qiáng)度、牽拉5 min即可引起TSCs立早基因c-fos m RNA表達(dá)的改變,牽拉30 min時(shí)c-fos m RNA相對(duì)表達(dá)量達(dá)到峰值。分化標(biāo)志基因表達(dá)在不同強(qiáng)度牽伸載荷下發(fā)生表達(dá)差異的時(shí)間點(diǎn)不同。說明牽伸載荷在早期對(duì)TSCs基因表達(dá)的影響具有時(shí)間和強(qiáng)度依賴性。
[Abstract]:Background: tendon injury is a micro tissue injury exists widely in athletes and ordinary workers, often occur repeatedly, after stretching the tendon, tendon showed local pain, swelling and limitation of movement function, histology showed tendon fibers, tendon rupture. Micro damage can not effectively repair is believed to induce tendon degeneration, mainly causes of disease. It has been reported that tendon rotator cuff tendon disease in the general population prevalence rate of 2.4~20% in high level athletes, Achilles tendon disease incidence was as high as 52%, competitive states to the everyday lives of the people and athletes had a serious impact. Therefore, to study the effect of micro damage repair factors, looking for promotion micro damage repair method is the problem to be solved. As the tendon tissue mechanical conduction between muscle and bone, maintain the physiological function of tendon stretchloading play an important role in some scholars. That long stretch over the tendon, the tendon injury can effectively repair is an important cause of tendon disease. Studies also have found that the centrifugal drafting training can relieve or cure tendon disease. Thus, stretchloading as a double-edged sword, can lead to tendon injury, and can promote tendon repair. So, study on Mechanism of traction load on the tendon, play its role, to avoid its adverse effects, is a reliable way to promote repair of tendon damage. Objective: 1. to investigate various stretchloading conditions on rat damage effect of Achilles tendon repair, clear to tendon repair the stress intensity, clarify the ideal effect on the biological stretchloading the characteristics of TSCs.2. to clarify the impact of early stretchloading early riser on the gene expression of TSCs. Methods: to establish the animal model of 1. rat Achilles tendon injury will only micro anesthesia in 6 8 week old male SD rats The right Achilles tendon, injection of collagenase solution as the experimental group, the left Achilles tendon PBS injection as the control group. 1 weeks after injection by the gross appearance of Achilles tendon, study performance and expression of differentiation marker gene compared with histopathology, determine the success of modeling.2. stretchloading on rats with micro damage repair effects of tendon 72 SD through the adaptability of rats after treadmill training, Achilles tendon injection of type I collagenase solution, preparation of micro Achilles tendon injury rat model. Then based on the treadmill strength condition of rats were randomly divided into 3 groups: control group (feeding cage), low intensity group (13m/min, 20min/day), the high intensity group (17m/min, 1h/day). On the treadmill at the start of the treadmill for 1 weeks treadmill at 4 weeks of rat Achilles tendon were observed the gross changes, pathological changes, biomechanical changes and tendon differentiation marker gene expression changes. To observe the effects of different running conditions of traction load on the intensity of micro Effect of injury of Achilles tendon repair, clear to the Achilles tendon repair ideal draft load intensity and time point. Then the ideal draft load intensity and time point, the experimental intervention of 9 8 week old male SD rats, and TSCs markers for extraction were: the control group (with the tendon of injection of PBS solution) the induction group (Achilles, injection of type I collagenase solution), induced by treadmill group (ideal strength + Achilles tendon type I injection collagenase solution + treadmill). To observe the intervention of biological effect on TSCs: crystal violet staining was used to observe the colony number; observation of colony morphology under inverted microscope; CCK8 method was used to observe the proliferation; flow cytometry to observe the cell apoptosis and cell surface marker; immunofluorescence dry marker; cytological staining, observe effects of RT-PCR cell differentiation in vitro stretchloading changes of.3. on rat Achilles tendon TSCs immediately early gene expression using this topic Group TSCs separation, mature culture, passage, cells using self-made uniaxial cyclic stretch device for in vitro cell traction. The 2%~12% were used to pull pull time grouping of cells and different strength hold, the use of cell RT-PCR was observed in the early stage of the distraction of c-fos gene, differentiation marker gene expression characteristics. Results: the rat Achilles tendon tissue compared with the control group, type 1.I collagenase induced, gross observation showed paratendon tissue increased, the lack of luster. Histological dull tendon showed increase of abnormal cells, disorder of collagen fibers, fiber tissue into tendon injury. The marker genes (Col I, TNMD (P0.05) expression was significantly reduced), osteogenic differentiation marker gene (Runx2) expression increased (P0.05).2. on the micro tendon injury model of rats after different intensity 1 weeks treadmill load stimulation, observe different groups of rat tendon in the large body, tissue pathology The biomechanical aspects and no visible changes (P0.05), but the low intensity group into the Achilles tendon tendon differentiation marker gene (Col I TNMD) expression were higher than that of control group and high intensity group (P0.05). After 4 weeks treadmill stretchloading stimulation, there is obvious difference between the experimental group: gross observation, low intensity group paratendon hyperplasia than in the control group and high intensity group decreased significantly; histological observation, semi quantitative study of low intensity group were significantly lower than the control group and high intensity group, and the high intensity group the histological score was significantly higher than the control group and the low intensity group (P0.05); Biomechanics the test, compared with the low intensity group and the control group, the ultimate stress and tensile strength were increased significantly (P0.05); low intensity group organized into tendon marker gene Col I compared with the control group and high intensity group increased significantly (P0.05), group TNMD: relative gene expression was not obvious 宸紓(p0.05).3.璇卞+璺戝彴緇勭殑TSCs緇嗚優(yōu)闆嗚惤鏁拌緝璇卞緇勫噺灝，

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