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目的探讨大鼠软骨非全层损伤模型的制备及术后细胞变化及细胞活化与整合素β_1表达的关系。方法 10周龄SD雄性大鼠45只,体质量300~400 g,随机分为手术组、假手术组和对照组,每组15只。手术组采用划痕法制造软骨非全层损伤模型;假手术组打开膝关节囊后直接缝合;对照组不行手术。术后1、7、14 d各组分别处死5只大鼠,取材行大体观察;并行HE、番红O组织学染色,评估并比较各组HE染色改良组织学评分及番红O染色灰度值;行Brd U、CD105免疫荧光染色和CD105/整合素β_1双标记染色,采用标准双盲法计数并比较各组各时间点CD105阳性细胞数。结果大体观察示手术组划痕周围软骨欠光滑,非透明,有软骨软化、纤维化改变,且随造模时间延长逐渐加重;假手术组和对照组软骨呈白色透明状,无软化、纤维化改变。HE染色示手术组在划痕周围细胞数增多,大小不等,排列分布不均;假手术组和对照组细胞大小均一,染色均匀,排列有序。手术组术后各时间点HE染色改良组织学评分均显著高于假手术组及对照组(P<0.05),各组组内各时间点间比较差异均无统计学意义(P>0.05)。番红O染色示手术组各时间点染色欠均匀,划痕周围区域着色较浅;假手术组和对照组各时间点染色均匀,无失染。术后各时间点各组间比较以及各组内各时间点间比较番红O染色灰度值,差异均无统计学意义(P>0.05)。Brd U免疫荧光染色示手术组划痕周围细胞排列紊乱,分布不均;假手术组和对照组细胞排列分布均匀,无聚集现象。CD105免疫荧光染色示手术组划痕周围有较多阳性细胞聚集,细胞大小不一、分布不均;假手术组和对照组软骨层见少数阳性细胞,分布均匀。手术组术后各时间点CD105阳性细胞数均显著多于假手术组和对照组(P<0.05);手术组内随时间延长CD105阳性细胞数逐渐增多,各时间点间比较差异均有统计学意义(P<0.05)。CD105/整合素β_1双荧光标记染色示手术组划痕周围有双标阳性细胞聚集,假手术组和对照组各时间点均未观察到双标阳性细胞。结论通过划痕能够建立大鼠软骨非全层损伤模型,且损伤无法完全修复。划痕造成的软骨损伤周围存在活化细胞聚集现象,细胞的活化与软骨基质改变关系不大;这些活化细胞处于增殖活跃状态,可同时表达CD105和整合素β_1。
Objective To investigate the preparation of non-full-thickness rat cartilage injury model and the relationship between the cell changes and the activation of cells and the expression of integrin β 1. Methods Forty-five SD male rats aged 10 weeks were randomly divided into operation group, sham operation group and control group with 15 rats in each group. In the operation group, the non-full-thickness cartilage injury model was made by the scratch method. In the sham operation group, the knee joint capsule was directly sutured and the control group was not surgically operated. At 1, 7 and 14 days after operation, 5 rats were sacrificed in each group, and the rats were randomly divided into three groups: the rats in each group were subjected to HE staining and Safranin O staining. The histological scores of HE staining and the safari O staining gray scale Values BrdU, CD105 immunofluorescence staining and CD105 / integrin β_1 double staining, using standard double-blind counting and comparison of each group at each time point CD105 positive cells. Results The general observation showed that the cartilage around the scars in the operation group was less smooth and non-transparent, with cartilage softening and fibrosis. The cartilage in the sham operation group and the control group was white and transparent without softening and fibrosis change. Hematoxylin and eosin staining showed that the number of cells around the scratch increased, the size was uneven, and the arrangement was unevenly distributed. The sham operation group and the control group had uniform cell size, uniform dyeing and orderly arrangement. The histological scores of HE staining at each time point in the operation group were significantly higher than those in the sham operation group and the control group (P <0.05). There was no significant difference between the time points in each group (P> 0.05). Safranin O staining showed that the staining of the operation group was not uniform at all time points, and the shades of the area around the scratch were lighter; the sham-operated group and the control group were evenly stained at every time point without any staining. There were no significant differences in the safranin O staining gray scales between the groups at various time points after operation and at any time point in each group (P> 0.05). BrdU immunofluorescence staining showed that the cells around the scratches in the operation group were disorderly arranged and distributed unevenly. The cells in the sham operation group and the control group were evenly distributed and had no aggregation. CD105 immunofluorescence staining showed that there were more positive cells around the scratches in the operation group, with different cell sizes and uneven distribution. In the sham operation group and the control group, a few positive cells were found in the cartilage layer, which were evenly distributed. The number of CD105 positive cells in the operation group was significantly higher than that in the sham operation group and the control group at each time point after operation (P <0.05). The number of CD105 positive cells in the operation group increased gradually over time, and there were statistically significant differences among the time points Significance (P <0.05). Double labeling of CD105 / integrin β_1 fluorescent staining showed that double positive cells were clustered around the scratches in the operation group. Double positive cells were not observed in sham operation group and control group at each time point. Conclusion Scratches can establish rat cartilage non-full-thickness injury model, and the injury can not be completely repaired. Scratches caused by cartilage injury around the existence of activated cell aggregation phenomenon, cell activation and cartilage matrix change is not significant; these activated cells in a proliferative state, which can express both CD105 and integrin β_1.