Gait analysis combined with the expression of TGF-β1, TGF-β3 and CREB during Achilles tendon healing in rat.
Gait analysis combined with the expression of TGF-β1, TGF-β3 and CREB during Achilles tendon healing in rat.
复制标题
步态分析与大鼠肌腱愈合过程中TGF-β1，TGF-β3和Creb的表达相结合。
DOI：
10.1016/j.cjtee.2021.10.002
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发表时间：
2021-11
期刊：
影响因子：
--
通讯作者：
Xiong Y
中科院分区：
文献类型：
--
作者：
Wu LM;Wang JK;Liu J;Fan CC;Wang YJ;Xiong Y
目的：观察大鼠跟腱愈合过程中步态行为及创面愈合因子转化生长因子-β-1、转化生长因子-β-3和cAMP反应元件结合蛋白-1(cAMP-β-1)的表达变化，探讨步态分析能否作为评价跟腱愈合的指标。将40只健康雄性SD大鼠的跟腱切断缝合，建立跟腱损伤(ATI)模型。按伤后1，2，4，6周观察时间点随机分为4组，每组10只。造模前，随机选择9只大鼠进行步态分析，包括步态周期、单步站立时间和平均速度。将数据记录为正常对照。此后，40只大鼠左侧后肢均建立ATI模型(ATI组)，右侧后肢仅切断缝合，不损伤跟腱(假手术组)。分别于伤后1、2、4、6周通过T台观察记录相应组大鼠(n=9)的步态行为。然后处死大鼠，取双侧跟腱。大体解剖和组织学观察肌腱愈合情况，免疫组织化学和定量聚合酶链式反应观察肌腱愈合过程中转化生长因子-β-1、转化生长因子-β-3、CREB-1蛋白和基因的表达。肌腱大体分级结果用Wilcoxon秩和检验进行分析，其余数据用多组间的单因素方差分析进行分析。与正常对照组相比，ATI后所有步态指标(步态周期、单步站立时间和平均速度)均受到较大影响，但随着时间的延长步态指标有所改善。步态周期在伤后1、2、4周显著低于正常对照组(与正常对照组比较，均P<0.05)，但在伤后6周基本恢复到正常水平((0.694±0.102)比(0.503±0.094)S，P<0.05)。针刺组术后1周、2周单立时间明显缩短(1周为(0.078±0.010)S，2周为(0.078±0.020)S，均为P<0.001)，4周时差异无统计学意义(P=0.120)。ATI组1、2、4、6周的平均速度明显低于正常对照组(P均<0.001)。大体观察显示，损伤后2、4、6周局部瘢痕粘连程度明显高于假手术组(均P<0.001)。ATI后6周形成广泛粘连。HE染色结果显示，伤后1、2、4周成纤维细胞数量逐渐增多，排列更整齐(P均<0.001)，6周时减少，但仍明显高于假手术组(P<0.001)。免疫组织化学结果显示，急性脑损伤组转化生长因子-β1、转化生长因子-β3、CREB-1的阳性表达在4个时间点均高于假手术组(P均<0.05)，术后2周达高峰，4周下降(分别为P=0.002，P=0.001，P=0.041)。定量聚合酶链式反应结果显示，急性脑损伤组各时间点转化生长因子-β-1、转化生长因子-β-3、CREB-1mRNA的表达均高于假手术组(均P<0.001)，术后2周达高峰，4周下降，6周明显下降(均P<0.05)。步态行为指数与跟腱愈合有关。本研究揭示了转化生长因子-β-1、转化生长因子-β-3、CREB-1在跟腱愈合过程中的变化，这些细胞因子可能在跟腱愈合过程中发挥调节作用。
To observe the changes of gait behavior and the expression of wound healing factors of transforming growth factor-β1 (TGF-β1), TGF-β3 and cAMP response element binding protein-1 (CREB-1) during the healing of Achilles tendon in a rat model, and to investigate whether gait analysis can be used to evaluate the tendon healing. Achilles tendon of 40 healthy male Sprague-Dawley rats were transected and sutured to establish the Achilles tendon injury (ATI) model. They were randomly divided into 4 groups based on the observational time point at 1, 2, 4 and 6 weeks after injury (n = 10 for each group). Before modeling, 9 rats were randomly selected for CatWalk gait analysis, which contained step cycle, single stance time and average speed. Data were recorded as the normal controls. After then, ATI models were established in the left hind limbs of the all 40 rats (ATI group), while the right hind limbs were only cut and sutured without injury of the Achilles tendon (sham operation group). At 1, 2, 4 and 6 weeks after injury, the gait behavior of the corresponding group of rats (n = 9) as observed and recorded by CatWalk platform. After then, the rats were sacrificed and Achilles tendon of both limbs was harvested. The tendon healing was observed by gross anatomy and histological examination, and the protein and mRNA expression of TGF-β1, TGF-β3, CREB-1 were observed by immunohistochemistry and qPCR. The results of tendon gross grading were analyzed by Wilcoxon rank sum test, and other data were analyzed by one-way analysis of variance among multiple groups. Compared with normal controls, all gait indexes (step cycle, single stance time and average speed) were greatly affected following ATI, which however improved with time. The step cycle was significantly lower at 1, 2 and 4 weeks after ATI (compared with normal controls, all p < 0.05), but almost returned to the normal level at 6 weeks ((0.694 ± 0.102) vs. (0.503 ± 0.094) s, p > 0.05). The single stance time of the ATI group was significantly shorter at 1 and 2 weeks after operation ((0.078 ± 0.010) s at 1 week, (0.078 ± 0.020) s at 2 weeks, all p < 0.001) and revealed no significant difference at 4 weeks (p = 0.120). The average speed of ATI group at 1, 2, 4, 6 weeks was significantly lower than that in the normal control group (all p < 0.001). Gross observation showed that the grade of local scar adhesion in ATI group increased significantly at 2, 4 and 6 weeks, compared with the sham operation group (all p < 0.001). Extensive adhesion was formed at 6 weeks after ATI. The results of HE staining showed that the number of fibroblast increased gradually and arranged more orderly in ATI group at 1, 2 and 4 weeks (all p < 0.001), and decreased at 6 weeks, but it was still significantly higher than that of the sham operation group (p < 0.001). Immunohistochemistry showed that the positive expression of TGF-β1, TGF-β3, CREB-1 in ATI group was higher than that in the sham operation group at 4 time points (all p < 0.05), which reached the peak at 2 weeks after operation and decreased at 4 weeks (p = 0.002, p < 0.001, p = 0.041, respectively). The results of qPCR suggested that the mRNA expression of TGF-β1, TGF-β3, CREB-1 in ATI group was higher than that in the sham operation group at all-time points (all p < 0.05), which reached the peak at 2 weeks after operation, decreased at 4 weeks, and significantly decreased at 6 weeks (all p < 0.001). Gait behavior indexes are associated with Achilles tendon healing. The study gives an insight of TGF-β1, TGF-β3, CREB-1 changes in the coursing of Achilles tendon healing and these cytokines may be able to be used to regulate the Achilles tendon healing.
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