核心蛋白聚糖对兔屈趾肌腱损伤位点胶原纤维形成以及成纤维细胞增殖的延迟效应

Delayed effects of decorin on the formation of collagen fibers and the proliferation of fibroblasts at the injured site of the flexor tendon of toes in rabbits

目的:观察兔屈肌腱损伤位点直接注射核心蛋白聚糖后对胶原纤维形成的作用,以及其改善兔屈肌腱损伤后的愈合效果。方法:实验于2004-09/2005-04在解放军第三军医大学高原军事医学系中心实验室进行。选取成年日本大耳白兔18只。按术后不同时间随机分成2,4,8周组,每组6只。①取兔后肢的第2趾,暴露深屈肌腱,横行切断。右侧肌腱缝合位点直接注射核心蛋白聚糖100μL(0.25g/L)为实验趾;左侧肌腱缝合位点注射磷酸盐缓冲液(1倍的磷酸盐缓冲液)100μL为对照趾。石膏固定。②各组兔肌腱大体粘连性状观察:于术后2,4,8周分别暴露粘连组织、腱鞘和肌腱,观察肌腱粘连形状(肌腱粘连分为4级,I级为无粘连,V级为广泛粘连)。③各组兔肌腱缝合段组织学观察:于术后2,4,8周取实验趾和对照趾肌腱缝合区切片。随机取2个标本切片做苏木精-伊红染色观察成纤维细胞计数;2个标本切片做Masson3色染色观察胶原纤维含量;2个标本行透射电镜观察肌腱愈合中成纤维细胞及胶原纤维的超微结构。结果:18只兔均进入结果分析。①兔肌腱大体粘连性状观察结果:8周组,实验趾吻合口光滑,愈合良好,Ⅰ,Ⅱ级粘连,肌腱滑动性好;对照趾吻合口形成Ⅳ,Ⅴ级粘连,与腱鞘很难分离,肌腱滑动性极差。②兔肌腱缝合段成纤维细胞计数结果:2周组实验趾显著少于对照趾[(708.67±73.30),(4289.33±55.79)个/视野]。4周组实验趾多于对照趾[(6734.83±192.91),(3322.33±183.32)个/视野]。8周组实验趾和对照趾无明显差异[(3525.17±166.36),(3267.50±167.91)个/视野]。③兔肌腱缝合段胶原纤维含量观察结果:光镜下观察,8周组实验趾胶原纤维含量明显增多,排列规则,胶原束直径一致,胶原纤维成熟。对照趾胶原纤维不规则,胶原束直径大小不一,成熟度有所增加。④兔肌腱缝合段超微结构观察结果:8周组实验趾胶原原纤维成熟排列规则,粗细均匀,纤维周期横纹清晰,腱细胞细胞质空泡减少。对照趾胶原原纤维不排列,纤维周期横纹不清晰,腱细胞活跃,胞突增多,胞质内大量空泡。结论:①核心蛋白聚糖对胶原的形成和成熟起到一个调节作用,使胶原束的直径更加一致,达到一个理想的胶原愈合。②延迟成纤维细胞的增殖,能够明显改善肌腱损伤后的愈合质量。

AIM：To observe the effect of decorin, directly injected to the injured site of rabbit＇s flexor tendons, on the formation of collagen fibers so as to improve the healing effect of injured flexor tendons. METHODS： The experiment was done in the Central Laboratory, College of High Altitude Military Medicine, Third Military Medical University of Chinese PLA ,from September 2004 to April 2005. Eighteen Japanese flapeared rabbits were randomized into 2-, 4- and 8-week groups with 6 ones in each group,①Second toes were obtained from rabbits＇ hind limbs to expose deep flexor tendon which was cross-cut. Right tendon of second toes used as experimental toes were directly injected with 100μL decorin （0.25 g/L） at the sutured site, and left tendon of second toes as control toes were injected with 100μL phosphat buffer solution. Plaster was used for fixation and immobilization.②Adhesion observation： Adherent tissues, tendon sheaths and tendons were exposed to observe the adherent shape of tendons at 2, 4 and 8 weeks after operation. There are four grades of tendon adhesion, grade 1 as no adhesion, V 02 as massive adhesion,③ Histological observation： Six sections were obtained respectively from the sutured region of experimental toes and control toes at 2, 4 and g weeks after operation. Two sections were selected randomly to perform hematoxylin and eosin （HE）staining so as to count the amount of fibroblasts. Another two sections were stained with Masson trichrome staining to observe the content of collagen fibers. The last two sections were observed under photoscape to study the ultra-structure of fibroblasts and collagen fibers in the healing of tendons. RESULTS： All the lg rabbits entered the result analysis.①General adhesive observation： In the 8-week group, stoma of experimental toes was smooth, and healing was good with Ⅰ,Ⅱ adhesion, and tendons had a good sliding feature. Ⅳ, Vadhesion was formed in the stoma of control toes, which was different to separate from tendon sheath, and it was very different for tendon to slide.② Cell counting： In the 2-week group, the number of fibroblasts in the experimental toes was significantly lower than that in the control toes [（708.67±73.30） vs.（4 289.33±55.79）per visual field]. In the ;4-week group, the number of fibroblasts in the experimental toes was higher than that in the control toes [（6 734.83±192.91） vs. （3 322.33±183.32）per visual field]. In the 8-week group, there was no significant difference in the number of fibroblasts between the experimental toes and control toes [（3 525.17±166.36）vs.（3 267.50±167.91）per visual field].③Content of collagen fibers： In the 8-week group, the content of collagen fibers was increased in the experimental toes under electron microscope, and the fibers arranged regularly and had the unified diameter, indicating that collagen fibers became mature. The fibers in the control group arranged irregularly, and had different diameters, indicating that an improvement in maturity.④ Uhrastructure observation： In the 8-week group, collagen fibers in the experimental toes became mature and arranged regularly with clear striae in fiber cycle. A decrease in the number of vacuoles in cytoplasm of tendon cells was found. However, the collagen fibers in the control toes arranged irregularly with unclear striae in fiber cycle, tendon cells became active, an increase in the number ofcell processes and a great amount of vacuoles in cytoplasm of tendon cells were found, CONCLUSION： Decofin plays a role in regulating the form and maturity of collagen, and unify the diameter of collagen bands so as to gain an ideal healing of tendon. Decorin also can delay the proliferation of fibroblasts, and dramatically improve the healing of the injured tendons.