AChE在小鼠皮肤切创愈合过程中的时序性表达及分布

Time-dependent Expression and Distribution of AChE during the Skin Incised Wound Healing in Mice

目的检测乙酰胆碱酯酶(acetylcholinesterase,AChE)在小鼠皮肤切创愈合过程中的时序性表达及分布特征,探讨其在创口愈合中的作用及其作为创口形成时间推断参考指标的可行性。方法 45只C57BL/KsJ小鼠被随机分为1个对照组和8个切创组。切创组建立皮肤切创模型,分别于伤后6h、12h、1d、3d、5d、7d、10d、14d提取皮肤创口样本;对照组提取未切创的皮肤组织。应用免疫组织化学染色、双重免疫荧光染色及Western印迹法检测皮肤样本中AChE的表达与分布。结果免疫组织化学染色结果显示,伤后6～12h,AChE主要表达于浸润的多形核白细胞;伤后1～3d,大量浸润的单个核细胞呈现AChE阳性染色;伤后5～14d,AChE阳性细胞以成纤维样细胞为主。AChE的阳性细胞率于伤后6h开始增多,伤后1d达到峰值。双重免疫荧光染色结果显示,AChE阳性的单个核细胞和成纤维样细胞主要为巨噬细胞和肌成纤维细胞。Western印迹法检测结果显示,各切创组AChE表达的相对表达水平均高于对照组,于伤后1d达到峰值,此后下降,于伤后7d达到第2个峰值。结论皮肤创口愈合期间AChE时序性表达于多形核白细胞、巨噬细胞和肌成纤维细胞,提示其可能参与创口愈合的炎症反应和纤维性修复的调节。联合使用多种方法检测AChE的表达,可以为创口形成时间的推断提供参考依据。

Objective To study the time-dependent expression and distribution of acetylcholinesterase (AChE) during skin incised wound healing in mice,and discuss its effect in wound healing as well as the feasibility of using it as a reference index for wound age estimation. Methods A total of 45 C57BL/KsJ mice were randomly divided into one control group and eight incised groups. The skin incised wound model was established in the incised groups with samples of skin wounds taken at 6 h, 12 h,1 d,3 d,5 d,7 d,10 d and 14 d post-injury respectively,while the uninjured skin tissue was extracted in the control group. Expression and distribution of AChE in skin samples were detected by immunohistochemistry, double immunofluorescence and Western blotting. Results Immunohistochemistry results indicated that AChE was mainly detected in infiltrating polymorphonuclear cells(PMNs)6 to 12 h post-injury. A large number of AChE-positive mononuclear cells (MNCs) were observed 1 to 3 d post-injury. The AChE-positive cells were mainly fibroblastic cells (FBCs) 5 to 14 d post-injury. The ratio of the AChE-positive cells increased initially 6 h post-injury,and reached the peak at 1 d post-injury. Double immunofluorescent staining showed that the majority of AChE-positive MNCs and FBCs expressed macrophage marker and myofibroblast marker,respectively. Western blotting results showed that the relative expression level of AChE in the incised group was higher than that in the control group averagely,reached the peak at 1 d post-injury,then reached a second peak at 7 d post-injury. Conclusion The expression of AChE is found in PMNs,macrophages and myofibroblast during skin wound healing,which indicates it might be involved in the adjustment of inflammatory response and fibrotic repair after injury. Moreover,combined use of various methods for the detection of the expression of AChE would provide reference for skin wound age estimation.