肌腱愈合早期的细胞凋亡和增殖及其分子机制变化

目的 探讨肌腱损伤后愈合早期细胞凋亡和增殖及相关控制和调控因子的变化.方法 采用15只来亨鸡,横断舣侧长趾浅、深屈肌腱,后行改良Kessler法修复.分别于术后3、7、14和28 d取趾深屈肌腱,以6根正常深屈肌腱作为0d对照组.通过TUNEL法榆测腱细胞凋亡,免疫荧光化学法检测增殖细胞核抗原(proliferation cell nuclear antigen,PCNA)和凋亡抑制蛋白bcl-2的表达,显微镜下计数荧光阳性细胞并统计分析,同时切片行苏木素染色观察组织反应和计数细胞总数.结果 (1)肌腱术后3、7、14、28 d细胞凋亡均显著高于0 d对照组(P＜0.05),其中腱内膜区3 d凋亡细胞数最多,14 d出现明显下降;而外膜区术后各时间点之间差异无统计学意义.(2)术后7 d细胞的PCNA蛋白表达较0 d对照组显著上升(P＜0.05),14 d达到高峰,28 d和正常水平差异无统计学意义.(3)bcl-2的变化趋势与PCNA相同.(4)组织炎症反应术后1周内最明显,14 d和28 d基本消失.结论 肌腱损伤3 d时细胞大量凋亡,增殖细胞很少;细胞增殖在损伤14 d时达到高峰,凋亡下降.bcl-2表达在14 d时达高峰,28 d时降至正常水平.

Molecular events of cellular apoptosis and proliferation in the early tendon healing period

Objective To study the molecular changes associated with apoptosis and proliferation of the tenocytes of the injured tendon in early healing period.Methods Fifteen Leghorn chickens were divid-ed into 5 groups.Flexor tendons of the long toes were transected and were repaired with modified Kessler method.The tendons were harvested 3,7,14 and 28 days postoperatively.Six normal flexor tendons were harvested as the controls.The apoptosis of tenocytes were quantified with the TUNEL assay.Protein levels of proliferation cell nuclear antigen(PCNA)and bcl-2 were assessed by immunofluorescence staining.The posi-tively labeled tenocytes were counted microscopically and compared statistically.We also stained sections with hematoxylin to observe their inflammation and count the total cells.Results 1)TUNEL assay showed that apoptotic cells of the tendons were significantly more in the injured tendons than those in the control(P＜0.05).The number of the apoptotic cells peaked on the 3rd day.2)Expression of the PCNA significantly in-creased on the 7th day(P＜0.05)and reached the peak on the 14th day.3)The pattern of changes in bcl-2expression was similar to that of PCNA.4)Tendon inflammatory changes were the most prominent in the first week.Inflammations disappeared gradually during the 2nd and 4th week.Conclusion The apoptosis in the tendon reaches the peak and at day 3 after injury with fewer proliferative cells.The proliferation peaks on the day 14 with significantly decreased apoptosis of tenocytes.The bcl-2 expression peaks at day 14 and de-clines to the normal levels at day 28.