Abstract：
The aim of the study was to explore the effect and mechanism of a low-level laser on hair follicle stem cells in full-thickness skin wound healing in mice. Full-thickness skin defects were generated by a 5-mm punch biopsy tool on the backs of depilated C57/BL6N mice, which were randomly divided thereafter into a low-dose laser treatment group (LLLT-Low), a high-dose laser treatment group (LLLT-High), and a control group (control). From the day of modeling to the day before the skin samples were taken, the wound area and wound edge of the mice in the LLLT-Low and LLLT-High groups were irradiated with a laser comb every 24 h, and the energy density was 1 J/cm2 and 10 J/cm2, respectively. The control group was irradiated with an ordinary fluorescent lamp. At 0, 3, 5, 10, and 14 days after modeling, pictures of each wound were taken, and the percent wound closure was analyzed. At 3, 5, 10, and 14 days after modeling, the samples were observed by hematoxylin and eosin (HE) and immunofluorescence (IF) staining. Whole transcriptome sequencing (RNA-Seq) was performed on the samples on day 10. Gene Ontology (GO) analysis was performed, and the results were validated by Western blot analysis and enzyme-linked immunosorbent assay (ELISA). The analysis of the percent of wound closure showed that healing was accelerated (significantly from 5 to 10 days) in the LLLT-Low group, but there was no clear change in the LLLT-High group. HE staining showed that the LLLT-Low group had an increasing number of hair follicles and a tendency to migrate to the center of the wound. There was no significant increase in the number of hair follicles and no obvious migration in the LLLT-High group. Immunofluorescence staining showed that the total number of CK15 + hair follicle stem cells in the LLLT-Low group was higher than that in the control group and LLLT-High group at all time points. The number and farthest migration distance of CK15 + hair follicle stem cells increased significantly with time, and after 5 days, they were significantly higher than those in the control group and LLLT-High group. RNA-Seq and Western blot analysis showed that the expression of related genes in hair follicle stem cells, including CK15, in the LLLT-Low group was upregulated. GO analysis and ELISA showed that the expression of many cytokines, represented by IL34, in the LLLT-Low group was upregulated. Low-level laser treatment can promote the proliferation, differentiation, and migration of CK15 + hair follicle stem cells by upregulating the cytokine IL34, thereby promoting skin wound healing in mice.
摘要：
该研究的目的是探讨低水平激光对小鼠全层皮肤伤口愈合中毛囊干细胞的影响及其机制。在脱毛的C57/BL6N小鼠的背上，通过5mm的穿孔活检工具产生了全层皮肤缺损，此后随机分为低剂量激光治疗组（LLLT-Low），高剂量激光治疗组（LLLT-High），和对照组（对照）。从建模那天到采集皮肤样本的前一天，每24小时用激光梳照射LLLT-Low和LLLT-High组小鼠的伤口面积和伤口边缘，能量密度分别为1J/cm2和10J/cm2。对照组用普通荧光灯照射。在建模后0、3、5、10和14天，拍摄了每个伤口的照片，并分析伤口闭合百分比。在建模后3、5、10和14天，采用苏木精、伊红(HE)和免疫荧光(IF)染色观察。在第10天对样品进行全转录组测序(RNA-Seq)。进行基因本体论（GO）分析，并通过Westernblot分析和酶联免疫吸附试验（ELISA）验证结果。伤口闭合百分比的分析表明，LLLT-Low组的愈合加速（从5到10天明显），但LLLT-High组无明显变化.HE染色显示LLLT-Low组的毛囊数量增加，并且有向伤口中心迁移的趋势。LLLT-High组毛囊数量无显著增加,无明显迁移。免疫荧光染色显示，在所有时间点，LLLT-Low组的CK15+毛囊干细胞总数均高于对照组和LLLT-High组。CK15+毛囊干细胞的数量和最远迁移距离随时间显著增加,5天后,显著高于对照组和LLLT-High组。RNA-Seq和Westernblot分析显示毛囊干细胞中相关基因的表达,包括CK15在内,LLLT-Low组上调.GO分析和ELISA显示,多种细胞因子的表达,以IL34为代表，在LLLT-Low组中上调。低水平激光治疗可以促进增殖，分化,通过上调细胞因子IL34使CK15+毛囊干细胞迁移,从而促进小鼠皮肤创伤愈合。
