肌腱愈合过程中转化生长因子β1基因表达的变化

背景:近年来,生长因子在肌腱愈合与粘连形成中的作用受到关注,其中转化生长因子与组织粘连与瘢痕形成的关系更倍受重视.目的:观察兔屈趾肌腱Ⅱ区伤口愈合过程中转化生长因子β1基因表达的变化.设计:随机对照动物实验.单位:青岛大学医学院附属医院骨科.材料:选用清洁级成年新西兰大白兔60只,体质量4.0～4.5 kg,雌雄不拘,由青岛市实验动物中心提供.所有动物的左前肢作为实验侧,同一动物右前肢作为对照侧.按术后1,7,14,21,28和56 d 6个时间点进行观察,每个时间点10只.其中6只进行原位杂交实验,4只进行免疫组织化学染色.实验过程中对动物的处置均符合动物伦理学标准.方法:实验于2005-09/2006-07在青岛大学医学院附属医院动物实验中心完成.麻醉后将所有动物左前中趾Ⅱ区屈趾深肌腱切断并用使用标准Kessler缝合法修复,对照侧不进行干预.分别于术后1,7,14,21,28和56 d麻醉后处死动物,实验侧沿原切口切开皮肤,切取肌腱与腱鞘.对照侧采取相同措施.主要观察指标:将肌腱与腱鞘组织进行原位杂交和免疫组织化学染色,观察转化生长因子β1的表达情况.结果:纳入的60只动物全部进入结果分析.①原位杂交结果:实验侧肌腱损伤后1 d,转化生长因子β1 Mrna的表达明显升高,在肌腱损伤后的14～21 d,转化生长因子β1 Mrna的表达持续升高达到最高峰,28 d开始下降,56d时仍保持较高水平.修复部位周围的腱鞘组织转化生长因子β1 Mrna的表达水平更高,在相同时间点,腱鞘细胞内的转化生长因子β1 Mrna的表达均高于肌腱组织.对照侧肌腱组织和腱鞘内均存在着转化生长因子β1 Mrna的表达,但是水平较低.实验侧各时间点腱鞘与肌腱细胞转化生长因子β1 Mrna的表达明显高于对照组,差异有显著性意义(P ＜ 0.05).②免疫组织化学染色结果:实验侧动物转化生长因子β1蛋白信号的表达术后第1 天开始增加,14～21 d达到高峰,56 d仍保持较高的水平.对照侧动物存在转化生长因子β1蛋白信号的表达,但表达水平较低.结论:正常无损伤的肌腱和腱鞘细胞能产生转化生长因子β1,当肌腱损伤后,细胞因子被激活,增加的细胞因子主要由肌腱细胞与腱鞘细胞产生,与肌腱的内、外源性愈合机制是一致的.

Gene expression of transforming growth factor beta-1 in tendon healing

BACKGROUND: We have paid more attention on the effects of growth factors on tendon healing and adhesion formation, especially on the correlation of transforming growth factor with tissue adhesion and scar formation. OBJECTIVE: To investigate the expression of transforming growth factor beta-1 mRNA in the zone Ⅱ flexor tendon of wound-healing rabbit models. DESIGN: Randomized controlled animal study. SETTING: Department of Orthopaedics, Affiliated Hospital of Medical College, Qingdao University. MATERIALS: Sixty clean adult New Zealand white rabbits weighting 4.0-4.5 kg, of either sex, were provided by Qingdao Animal Experimental Center. Left forelimbs of each animal were as experimental side, and right forelimbs of each animal were as control. There were 6 time points, namely at days 1, 7, 14, 21, 28 and 56, 10 rabbits in each time point. Of the 10 rabbits, 6 rabbits received the in situ hybridization and 4 rabbits received the immunohistochemical staining. Animal intervention met the animal ethical standard. METHODS: Experiments were performed at the Animal Experimental Center of Hospital Affiliated to Medical College of Qingdao University from September 2005 to July 2006. After anesthesia, each rabbit underwent complete transection of the profundus middle flexor tendon in zone Ⅱ, and then the tendon was repaired by the Kessler method. Rabbits in the control group did not receive any intervention. Rabbits were anesthetized and killed 1, 7, 14, 21, 28 and 56 days after the surgery. Skin was incised along the original incision at the experimental sides to obtain tendons and tendon sheaths. The same measurements were performed in the control group. MAIN OUTCOME MEASURES: Tenocytes and tendon sheath cells were detected with the in situ hybridization and the immunohistochemical staining to observe the expression of transforming growth factor beta-1. RESULTS: Sixty rabbits were involved in the result analysis. ①The in situ hybridization results: Expression of transforming growth factor beta-1 mRNA was increased at day 1 after tendon injury in the experimental group, reached a peak at days 14-21 after tendon injury, reduced at day 28 and was still in a high level at day 56. Expression of transforming growth factor beta-1 mRNA was high in tendon sheath cells around the repaired region. At the same time point, the expression of transforming growth factor beta-1 mRNA was higher in tendon sheath cells than in tenocytes. Low expression of transforming growth factor beta-1 mRNA was found in tenocytes and tendon sheath cells in the control group. The expression of transforming growth factor beta-1 mRNA in tenocytes and tendon sheath cells was higher in the experimental group than in the control group at each time point (P ＜ 0.05). ②Immunohistochemical staining results: Expression of transforming growth factor beta-1 protein was elevated at day 1 after the surgery, reached the peak at days 14-21 and was still in a high level at day 56 in the experimental group. Low expression of transforming growth factor beta-1 protein was seen in the control group. CONCLUSION: The normal uninjured tenocytes and tendon sheath cells produce transforming growth factor beta-1. The cytokine is activated in the injured tendon. The increase of this cytokine in both tenocytes and tendon sheath fibroblasts are coincidence with both extrinsic and intrinsic mechanisms for tendon repair.