Chen L, Li J, Li J Y, Zhang E Y, Zhu Z Z
Division of Spine Surgery, Department of Orthopedic Surgery, Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing 210008, China Department of Plastic & Cosmetic Surgery, Women's Hospital of Nanjing Medical University (Nanjing Women and Children's Healthcare Hospital),Nanjing 210004, China.
Department of Plastic & Cosmetic Surgery, Women's Hospital of Nanjing Medical University (Nanjing Women and Children's Healthcare Hospital),Nanjing 210004, China.
Zhonghua Yu Fang Yi Xue Za Zhi. 2024 Sep 6;58(9):1397-1403. doi: 10.3760/cma.j.cn112150-20240109-00037.
This study aims to investigate the effects of the skin tissue derived peptides on proliferation, apoptosis, migration and collagen expressions in keloid fibroblasts. From January 2015 to January 2017, patients with hypertrophic scar who underwent surgical excision in department of plastic surgery of Nanjing maternal and child health hospital were included in this retrospective study. Four peptides were selected from the differential peptides between human hypertrophic scar and normal skin tissue. They were named as peptide deregulated in hypertrophic scar 2-5 (PDHPS2-5). Bioinformatics and functional analysis were performed. A low dose of 10 μmol/L of four peptides were respectively added to the culture medium of human primary keloid fibroblasts for 24 h. Cell counting kit-8 (CCK-8) were used to detect the changes in cell viability. Cell apoptosis was detected by flow cytometry. Cell migration ability was checked by Transwell chamber. The protein expressions of collagen COL1A2 (Collagen type I alpha 2) and the myofibroblast marker gene ACTA2 (Actin alpha 2) were analyzed by Western blot. The results showed that bioinformatics prediction analysis revealed that peptide PDHPS4 has the longest half-life and the highest thermal stability. Compared with the control group, low dose of four peptides had no significant effect on the survival rate and apoptosis of keloid fibroblasts tested by CCK-8 assay and flowcytometry. Transwell analysis showed that one peptides (PDHPS5) can significantly inhibit the cell migration ability (The optical density value in Control is 0.81±0.11, in PDHPS5 is 0.27±0.03, =8.61, =0.001). Western blot analysis showed that four peptides (PDHPS2, PDHPS3, PDHPS4, PDHPS5) can significantly inhibit the protein expressions of COL1A2 (The relative protein band intensity in Control is 1.02±0.02, in PDHPS2 is 0.21±0.04, in PDHPS3 is 0.26±0.03, in PDHPS4 is 0.53±0.04, in PDHPS5 is 0.73±0.04, =31.38, 38.54, 18.88, 11.07 respectively, all value are less than 0.01). Three peptides (PDHPS2, PDHPS3, PDHPS5) can significantly inhibit the protein expressions of ACTA2 (The relative protein band intensity in Control is 1.02±0.02, in PDHPS2 is 0.64±0.05, in PDHPS3 is 0.77±0.06, in PDHPS5 is 0.47±0.07, =12.08, 6.38, 14.06 respectively, all value are less than 0.01). In conclusion, the differentially expressed peptides in human hypertrophic scar tissue can affect the function of keloid fibroblasts and collagen expressions to varying degrees. Among them, two peptides (PDHPS2,PDHPS3) significantly inhibit the protein expressions of COL1A2 and ACTA2. The peptide PDHPS5 has high stability, significantly suppresses cell migration, and reduces the protein expressions of COL1A2 and ACTA2, which may provide a new strategy for scar prevention and treatment.
本研究旨在探讨皮肤组织衍生肽对瘢痕疙瘩成纤维细胞增殖、凋亡、迁移及胶原蛋白表达的影响。2015年1月至2017年1月，选取在南京市妇幼保健院整形外科接受手术切除的增生性瘢痕患者纳入本回顾性研究。从人增生性瘢痕与正常皮肤组织的差异肽中筛选出4种肽，命名为增生性瘢痕中失调肽2 - 5（PDHPS2 - 5）。进行生物信息学和功能分析。将低剂量10 μmol/L的4种肽分别加入人原代瘢痕疙瘩成纤维细胞的培养基中培养24小时。采用细胞计数试剂盒 - 8（CCK - 8）检测细胞活力变化。通过流式细胞术检测细胞凋亡。采用Transwell小室检测细胞迁移能力。通过蛋白质印迹法分析胶原蛋白COL1A2（I型胶原蛋白α2）和肌成纤维细胞标志物基因ACTA2（肌动蛋白α2）的蛋白表达。结果显示，生物信息学预测分析表明肽PDHPS4半衰期最长且热稳定性最高。与对照组相比，低剂量的4种肽经CCK - 8法检测和流式细胞术检测对瘢痕疙瘩成纤维细胞的存活率和凋亡无显著影响。Transwell分析显示，一种肽（PDHPS5）能显著抑制细胞迁移能力（对照组光密度值为0.81±0.11，PDHPS5组为0.27±0.03， =8.61， =0.001）。蛋白质印迹分析显示，4种肽（PDHPS2、PDHPS3、PDHPS4、PDHPS5）均能显著抑制COL1A2的蛋白表达（对照组相对蛋白条带强度为1.02±0.02，PDHPS2组为0.21±0.04，PDHPS3组为0.26±0.03，PDHPS4组为0.53±0.04，PDHPS5组为0.73±0.04，分别为31.38、38.54、18.88、11.07，所有 值均小于0.01）。3种肽（PDHPS2、PDHPS3、PDHPS5）能显著抑制ACTA2的蛋白表达（对照组相对蛋白条带强度为1.02±0.02，PDHPS2组为0.64±0.05，PDHPS3组为0.77±0.06，PDHPS5组为0.47±0.07，分别为12.08、6.38、14.06，所有 值均小于0.01）。综上所述，人增生性瘢痕组织中差异表达的肽可不同程度地影响瘢痕疙瘩成纤维细胞功能和胶原蛋白表达。其中，两种肽（PDHPS2、PDHPS3）显著抑制COL1A2和ACTA2的蛋白表达。肽PDHPS5具有高稳定性，能显著抑制细胞迁移，并降低COL1A2和ACTA2的蛋白表达，这可能为瘢痕防治提供新策略。
