成骨细胞特异性因子2及其相关因子在大鼠皮肤创面瘢痕重塑期中的表达变化及意义

Variations in expressions of periostin and related factors in early stage of wound healing and scarremodeling in rats

目的 观察成骨细胞特异性因子2（PN）、血管生成素-1（Ang-1）、血管内皮生长因子（VEGF）及其受体2（Flk-1）在创面及瘢痕中的表达情况,探究其对创伤愈合和瘢痕形成的意义.方法 雄性SD大鼠82只,按随机数字表法将大鼠按预定处死时间分组,每组8～9只,背中部脊柱旁左右各制备1个皮肤全层（正常皮肤）及皮下浅层组织的创面（2cm×2cm）,观察创面瘢痕并测量愈合面积；苏木素-伊红（HE）染色行组织病理学观察,免疫组化法检测4～8周创面瘢痕PN、Ang-1、VEGF、Flk-1的表达；各时间点用蛋白质免疫印迹试验（Western blotting）检测PN、Ang-1、VEGF的表达.以正常皮肤作为对照.结果 HE染色显示,4～8周创面组织已愈合、完成了上皮化.免疫组化结果显示,愈合创面组织中Flk-1表达量与正常皮肤无差异,PN表达量（A值/μm2）仅在伤后5周低于正常皮肤（2.43±0.44比4.24 ± 0.50,P＜0.05）,4、5、6、8周Ang-1与VEGF的表达量（A值/μm2）均低于正常皮肤（Ang-1：3.51±0.93、3.10 ±0.57、2.77±0.59、2.77±1.26比4.89±0.48,VEGF：1.76±0.68、1.75±0.49、1.99±0.42、1.94±0.86比4.86±1.63,均P＜0.05）；在创面瘢痕中,PN、Flk-1的阳性信号主要集中于细胞内,Ang-1、VEGF的阳性信号主要存在于细胞外基质.Western blotting结果显示,PN、Ang-1、VEGF表达均在创面10 d时达到高峰,并高于正常皮肤表达量的7.90 ～ 22.56倍（PN：2.45±1.51比0.31±0.19、Ang-1：18.43±15.20比1.53±1.42,VEGF：6.09±4.66比0.27±0.13,P＜0.05或P＜0.01）,随后迅速下降.结论 PN、Ang-1、VEGF、Flk-1在大鼠全层皮肤缺损创面愈合过程中存在一过性的表达升高,在创面瘢痕中的表达部位和局部表达量与创伤早期相比有一定区别；它们共同参与了大鼠全层皮肤缺损创面修复的过程,其作用可能在创伤修复过程的增生期,在瘢痕中的低表达可能与大鼠的无瘢痕愈合有关.

Objective To investigate the expressions of periostin （PN）,angiopoietin-1 （Ang-1）,vascular epithelial growth factor （VEGF） and fetal liver kinase-1 （Flk-1） during the processes of scar formation and modulation in rat cutaneous wounds and probe into their roles in wound healing and scaring.Methods Eighty-two male Sprague-Dawley （SD） rats were randomly divided into 10 groups with 8-9 rats in each group.Two 2 cm × 2 cm full-thickness excisional wounds in the back were created in each rat.The wound surface was observed,and the healing area was measured.The pathological change was observed after hematoxylin and eosin （HE） staining.The expressions ofPN,Ang-1,VEGF and Flk-1 in wound surface scar at 4-8 weeks were determined with immunohistochemistry.The expressions of PN,Ang-1 and VEGF were determined by Western blotting.The normal skin was served ais control.Results HE staining showed that the wound surface tissue had healed with epithelization at 4-8 weeks.Immunohistochemistry results showed that there was no significant difference in Flk-1 expression between wound surface tissue and normal skin.The PN expression （A value/μm2） in wound surface tissue was significantly lower than that in normal skin at 5 weeks （2.43 ± 0.44 vs.4.24 ± 0.50,P〈0.05）,and the expression of Ang-1 and VEGF （A value/lμm2） at 4,5,6,8 weeks was significantly lower than that in normal skin （Ang-1：3.51 ± 0.93,3.10 ± 0.57,2.77 ± 0.59,2.77 ± 1.26 vs.4.89 ± 0.48; VEGF：1.76 ±0.68,1.75 ±0.49,1.99 ±0.42,1.94 ±0.86 vs.4.86 ± 1.63,all P〈0.05）.In wound surface scar,PN and Flk-1 positive signal was found in cell,and the Ang-1 and VEGF positive signal in extracellular matrix.Western blotting data demonstrated that the expressions of PN,Ang-1 and VEGF peaked at the 10th day after excision with increases to 7.90-22.56 folds compared with normal skin （PN：2.45 ± 1.51 vs.0.31 ± 0.19,Ang-1：18.43 ± 15.20 vs.1.53 ± 1.42,VEGF：6.09 ± 4.66 vs.0.27 ± 0.13,P〈0.05 or P〈0.01）,and then followed with a decrease.Conclusions PN,Ang-1,VEGF and Flk-1 are transiently overexpressed in early stage of full-thickness cutaneous wound healing in rats.Their expressions vary in wounds and scars.They participate in the healing of full-thickness cutaneous wounds together and may be essential for the proliferation stage during wound healing.