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目的:通过体外对大鼠跟腱成纤维细胞过度拉伸来模拟体育训练跟腱损伤后重塑的效应分子MMP-2和TIMP-1,2的响应和相关信号通路。方法:原代分离大鼠跟腱成纤维细胞,扩增后采用等双轴细胞力学拉伸装置对细胞进行:1)正常对照组;2)14%力学拉伸组;3)力学拉伸组并加入NF-κB信号通路抑制剂。拉伸后提取细胞上清液进行明胶酶谱法和Western Blotting检测MMP-2以及NF-κB磷酸化蛋白;并提取细胞总RNA,反转录后进行半定量PCR对TIMP-1,2进行检测。,然后对MMP-2,TIMP-1,2等进行基因水平和蛋白水平的检测。结果:1)力学损伤可使跟腱成纤维细胞MMP-2表达明显上升(P<0.05);2)力学损伤对跟腱成纤维细胞中TIMP-1,2的表达无显著性影响;3)力学损伤可引起NF-κB磷酸化蛋白表达明显上升(p<0.05);4)NF-κB抑制剂可明显抑制力学损伤引起的MMP-2过量表达(p<0.05)。结论:体育运动引起的过度拉伸损伤可通过NF-κB上调MMP-2的表达和活性,提示过度拉伸引起的运动损伤可能与跟腱损伤和病变通过MMP直接相关。
OBJECTIVE: To simulate the response of MMP-2 and TIMP-1, and the related signaling pathways remodeled after traumatic injury in rats with tendon injury by over-stretching rat Achilles tendon fibroblasts in vitro. Methods: Primary rat Achilles tendon fibroblasts were isolated and expanded. The cells were subjected to isobaric mechanical stretching: 1) normal control group; 2) 14% mechanical stretching group; 3) mechanical stretching group And add NF-κB signaling inhibitors. The supernatant of the cells was extracted and the MMP-2 and NF-κB phosphorylation proteins were detected by gelatin zymography and Western Blotting. The total RNA was extracted and reverse transcribed to detect TIMP-1 and 2 . , Then MMP-2, TIMP-1, 2 and so on gene level and protein level detection. Results: 1) The mechanical damage could significantly increase the expression of MMP-2 in Achilles fibroblasts (P <0.05); 2) The mechanical damage had no significant effect on the expression of TIMP-1,2 in Achilles fibroblasts; 3) Mechanical damage can cause NF-κB phosphorylation protein expression was significantly increased (p <0.05); 4) NF-κB inhibitor can significantly inhibit mechanical damage caused by MMP-2 overexpression (p <0.05). CONCLUSION: Excessive stretching induced by exercise can up-regulate the expression and activity of MMP-2 by NF-κB, suggesting that exercise-induced over-stretching may be directly related to Achilles tendon injury and lesion through MMPs.