Collagen Ⅴ在推拿促进长期大负荷运动大鼠肌肉干细胞自我更新中的作用研究

The Role of Collagen Ⅴ in Tuina Promoting Self-Renewal of Muscle Satellite Cells Undergoing Long-Term Large Load Exercise in Rats

关键词:  长期大负荷运动  推拿  肌肉干细胞  自我更新  Ⅴ型胶原蛋白

Key words:  long-term large load exercise  Tuina  muscle satellite cells  self-renewal  Collagen Ⅴ

摘要:

目的：观察推拿对长期大负荷运动大鼠骨骼肌损伤修复、后肢抓力及Collagen Ⅴ表达的影响，探讨推拿促进大鼠肌肉干细胞自我更新的机制。方法：32只雄性SD大鼠随机分为空白对照组（C组）、长期大负荷运动组（E组）、长期大负荷运动+推拿组（ET组）、长期大负荷运动+假推拿组（ES组）。C组不做任何处理，E组、ET组和ES组进行4周大负荷跑台运动制备长期大负荷运动损伤模型；E组不予任何干预；ET组于每次运动后6h予以推拿干预；ES组于每次运动后6h仅予以抚摸干预。测定初始及4周后大鼠的体重和后肢抓力。末次干预后24h取大鼠两侧腓肠肌，采用透射电镜观察肌纤维超微结构变化；免疫组织化学染色检测Collagen Ⅴ表达水平；实时荧光定量PCR检测Col5a1和Col5a3 mRNA表达水平；Western Blot检测Pax7、MyoD和Myf5蛋白表达水平；双重免疫荧光染色检测Pax7与Collagen Ⅴ共定位。结果：与C组相比，E组、ES组和ET组大鼠后肢抓力增加值差异显著（P<0.01），E组和ES组超微结构均出现肌原纤维断裂等变化，E组和ES组Collagen Ⅴ、Pax7、MyoD、Myf5、Col5a1和Col5a3 mRNA以及Pax7与Collagen Ⅴ共定位表达显著降低（P<0.05）；与E组相比，ET组后肢抓力增加值差异显著（P<0.05），超微结构表现良好，Collagen Ⅴ、Pax7、MyoD、Myf5、Col5a3 mRNA以及Pax7与Collagen Ⅴ共定位表达显著升高（P<0.05）。结论：推拿可通过上调Collagen Ⅴ表达，促进大鼠骨骼肌肌肉干细胞自我更新，从而加速修复长期大负荷运动诱发的超微结构损伤，增强肌肉力量。

Abstract:

Objective: To observe the effects of Tuina on the repair of skeletal muscle injury, grasping force of hind limbs and expression of Collagen Ⅴ (COLⅤ) after long-term large load exercise in rats, and to explore the mechanism of Tuina promoting the self-renewal of muscle satellite cells (MSC). Methods: A total of 32 male Sprague-Dawley rats were randomly divided into groups C, E, ET, and ES. Group C did not do any treatment, while the other rats did large load treadmill exercise for 4 weeks to prepare gastrocnemius injury model. Group E received no intervention. Group ET received Tuina 6h after each exercise, while group ES only received stroke. Body weight and grasping force of hind limbs of the rats were measured at the beginning and 4 weeks later. after 24h of the last exercise and Tuina, samples of gastrocnemius were obtained from both sides of all the rats, and observed the ultrastructure of skeletal muscle fibers by transmission electron microscopy, detected the expression of Collagen Ⅴ with immunohistochemical staining, the expression of mRNA Col5a1 and Col5a3 with RT-PCR, and the expression of Pax7, MyoD and Myf5 protein with Western blotting, observed co-localization by labeled Pax7 and Collagen Ⅴ with Double immunofluorescence staining. Results: Compared with group C, the increased grasping force of hind limbs in group E, ES and ET significantly different (P<0.01), the changes such as myofibril fracture were observed in the ultrastructure of group E and ES, and the expression of Collagen Ⅴ, Pax7, MyoD, Myf5 protein, mRNA Col5a1, Col5a3 and the expressions of co-location of Pax7 and Collagen Ⅴ in group E and ES decreased significantly (P<0.05). Compared with group E, the increased grasping force of hind limbs in group ET significantly different (P<0.05), the expression of Collagen Ⅴ, Pax7, MyoD, Myf5 protein, mRNA Col5a1, Col5a3 and the expressions of co-location of Pax7 and Collagen Ⅴ in group ET increased significantly (P<0.05), and the ultrastructural damage recovered well. Conclusion: Tuina can promote the self-renewal of skeletal muscle satellite cells in rats by up-regulating expression of Collagen Ⅴ, to accelerate the repair of ultrastructural damage induced by long-term large load exercise and enhancing muscle strength.