牵伸载荷对大鼠跟腱微损伤的修复作用及其对TSCs立早基因的影响

发布时间：2018-03-30 15:10

  本文选题：肌腱微损伤　切入点：牵伸载荷　出处：《第三军医大学》2017年硕士论文

【摘要】：研究背景:肌腱微损伤是运动员和普通劳动者中广泛存在的组织损伤,常发生于反复、过度拉伸肌腱之后,表现为肌腱局部疼痛、肿胀和运动功能受限,组织学表现为肌腱纤维紊乱、断裂。肌腱微损伤不能有效修复被认为是诱发肌腱退变、肌腱病的主要原因。有报道显示肩袖腱病在普通人中发病率为2.4~20%,跟腱病在高水平运动员中发病率高达52%,这对人们的日常生活和运动员的竞技水平产生了严重的影响。因此,研究微损伤修复的影响因素,寻找促进微损伤修复方法是目前仍有待解决的问题。肌腱作为肌肉与骨之间的力学传导组织,牵伸载荷对肌腱生理功能的维持起到重要作用。有学者认为,长期过度牵拉肌腱,使肌腱的损伤无法有效修复是导致肌腱病的重要原因。同时也有研究发现,通过离心性牵伸训练可以缓解甚至治愈肌腱病。由此可见,牵伸载荷如一柄双刃剑,即可导致肌腱损伤,又能促进肌腱修复。所以,研究牵伸载荷对肌腱的作用机制,发挥其有利作用,避免其不利影响,是促进肌腱微损伤修复的可靠途径。目的:1.探讨不同牵伸载荷条件对大鼠微损伤跟腱修复的影响,明确有利于肌腱修复的应力强度,阐明理想牵伸载荷对TSCs生物学特性的影响。2.阐明牵伸载荷早期对TSCs立早基因表达的影响。方法:1.大鼠跟腱微损伤动物模型的建立将6只8周龄雄性SD大鼠麻醉后,右侧跟腱注射胶原酶溶液作为实验组,左侧跟腱注射PBS溶液作为对照组。注射1周后通过跟腱组织的大体表现、组织病理学表现和分化标志基因表达对比,判断造模是否成功。2.牵伸载荷对大鼠跟腱微损伤修复的影响将72只SD大鼠经过适应性跑台训练后,跟腱注射I型胶原酶溶液,制备跟腱微损伤大鼠模型。然后依据跑台强度条件将大鼠随机分为3组:对照组(笼中饲养)、低强度组(13m/min,20min/day)、高强度组(17m/min,1h/day)。在跑台开始时、跑台1周、跑台4周时分别观察大鼠跟腱的大体变化、组织病理学变化、生物力学变化以及成肌腱分化标志基因表达变化。观察不同跑台条件的牵伸载荷强度对微损伤跟腱修复的影响,明确有利于跟腱修复的理想牵伸载荷强度和时间点。然后利用该理想牵伸载荷强度和时间点,对9只8周龄SD雄性大鼠进行实验干预,并分别将提取的TSCs标记位为:对照组(跟腱注射PBS溶液)、诱导组(跟腱注射I型胶原酶溶液)、诱导+跑台组(跟腱注射I型胶原酶溶液+理想强度跑台)。观察干预对TSCs的生物学影响:结晶紫染色观察集落数量;倒置相差显微镜观察集落形态;CCK8法观察增殖能力;流式细胞学观察细胞凋亡和细胞表面标志物;免疫荧光观察干性标志物;细胞学染色、RT-PCR观察细胞分化能力变化。3.体外牵伸载荷对大鼠跟腱来源TSCs立早基因表达的影响沿用本课题组成熟的TSCs分离、培养、传代技术,利用自制细胞单轴循环牵拉装置在体外对细胞进行牵拉。分别用2%~12%的牵拉强度和不同牵拉时间对细胞进行分组,利用RT-PCR观察细胞在牵拉早期c-fos基因、分化标志基因表达的特点。结果:1.I型胶原酶溶液诱导的大鼠跟腱组织与对照组相比,大体观察表现为腱旁组织增多,肌腱黯淡缺乏光泽。组织学表现为异形细胞增多、胶原纤维排列紊乱、纤维损伤等。组织成肌腱标志基因(Col I、TNMD)表达明显减少(p0.05),成骨分化标志基因(Runx2)表达增多(p0.05)。2.对肌腱微损伤模型大鼠经过1周的不同强度跑台负荷刺激后,各实验组大鼠的跟腱在大体观察、组织病理学表现及生物力学表现等方面未观察到明显变化(p0.05),但低强度组跟腱的成肌腱分化标志基因(Col I、TNMD)相对表达量较对照组和高强度组均升高(p0.05)。经过4周跑台牵伸载荷刺激后,各实验组间出现明显差异:大体观察,低强度组腱旁增生组织较对照组和高强度组明显减少;组织学观察,低强度组组织学半定量评分显著低于对照组和高强度组,而高强度组的组织学评分明显高于对照组和低强度组(p0.05);生物力学测试,低强度组与对照组相比,最终应力与抗拉强度均明显升高(p0.05);低强度组的组织成肌腱标志基因Col I较对照组和高强度组明显升高(p0.05),各组间TNMD基因相对表达量未见明显差异(p0.05)。3.诱导+跑台组的TSCs细胞集落数较诱导组减少,集落中的细胞分部较诱导组更紧凑,CCK-8法检测到24 h和48 h的诱导+跑台组TSCs的OD值较诱导组增高(p0.05)。细胞凋亡实验在各实验组间无明显差异。诱导+跑台组TSCs的细胞干性标记物Nanog较诱导组表达增多(p0.05)。诱导+跑台组TSCs的细胞标志物中CD44、CD73比例较对照组和诱导组升高。诱导+跑台组TSCs的成骨分化标志基因(Runx2、Dlx5)、成软骨分化标志基因(Col II)相对表达量较诱导组明显减少(p0.05);而诱导+跑台组成肌腱分化标志基因(Col I、TNMD)、成脂分化标志基因(ap2、PPARγ)相对表达量较诱导组明显增多(p0.05)。经过成骨诱导培养后,诱导+跑台组的TSCs成骨分化标志基因(Runx2、Dlx5)相对表达量较对照组和诱导组显著升高(p0.05),诱导组的Runx2相对表达量较对照组明显降低(p0.05);经过成脂肪诱导培养后,诱导+跑台组TSCs的成脂肪分化标志基因(ap2、PPARγ)相对表达量较诱导组明显升高(p0.05),其中PPARγ相对表达量较对照组亦显著升高(p0.05),诱导组TSCs的PPARγ相对表达量较对照组显著降低(p0.05);经过成软骨诱导培养后,诱导+跑台组TSCs成软骨分化标志基因(Col II、Sox9)的相对表达量较诱导组明显降低(p0.05);而诱导组TSCs的Col II、Sox9基因相对表达量较对照组、诱导+跑台组均明显上升(p0.05)4.与对照组相比,TSCs在4%和8%牵拉强度下在单轴循环牵拉30 min时c-fos mRNA相对表达量达到峰值(p0.05)。2%牵拉强度即可使TSCs的c-fos m RNA相对表达量升高(p0.05),6%、8%、12%牵拉强度可使其相对表达量进一步升高(p0.05)。时间方面,仅牵拉5 min即可使c-fos相对表达量明显升高(p0.05)。8%牵拉强度在120 min时,Col I、TNMD、Dlx5、Runx2等基因相对表达量均升高(p0.05)。结论:1.I型胶原酶溶液注射入SD大鼠跟腱后能够在大体和组织病理学表现上模拟肌腱微损伤表现,该模型是一种比较理想的动物模型。2.不同强度牵伸载荷在1周对微损伤修复的影响无差异。低强度(13m/min,20min/day)跑台牵伸载荷4周能够促进大鼠微损伤跟腱的修复;高强度(17m/min,1h/day)跑台牵伸载荷4周阻碍微损伤肌腱的修复。因此对大鼠微损伤跟腱的修复来说,13m/min,20min/day跑台4周是较为理想的牵伸载荷条件。3.理想牵伸载荷条件对大鼠跟腱的TSCs影响主要表现在增殖能力强、集落形态集中、细胞干性增强、CD44和CD73表达增多、细胞向成肌腱和成脂肪方向分化等方面,说明牵伸载荷对TSCs增殖和分化具有明显的调控作用。4.体外单轴循环牵拉TSCs实验中,2%牵拉强度、牵拉5 min即可引起TSCs立早基因c-fos m RNA表达的改变,牵拉30 min时c-fos m RNA相对表达量达到峰值。分化标志基因表达在不同强度牵伸载荷下发生表达差异的时间点不同。说明牵伸载荷在早期对TSCs基因表达的影响具有时间和强度依赖性。
[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�寮�(p0.05).3.璇卞��+璺戝彴缁勭殑TSCs缁嗚優闆嗚惤鏁拌緝璇卞�肩粍鍑忓埃�

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