Zhang Q Y, Zhang L X, Han D H, Jiao X C, Zheng Z, Guo K, Yang Y S
Department of Burns and Cutaneous Surgery, Burn Center of PLA, the First Affiliated Hospital of Air Force Medical University, Xi'an 710032, China The Third Student Battalion, School of Basic Medical Sciences of Air Force Medical University, Xi'an 710032, China.
Department of Burns and Cutaneous Surgery, Burn Center of PLA, the First Affiliated Hospital of Air Force Medical University, Xi'an 710032, China.
Zhonghua Shao Shang Yu Chuang Mian Xiu Fu Za Zhi. 2023 Dec 20;39(12):1168-1174. doi: 10.3760/cma.j.cn501225-20231030-00154.
To explore the expression of endosialin, i.e., CD248 in human hypertrophic scars (HSs) and its regulatory effect on the phenotype of hypertrophic scar fibroblasts (HSFs). The method of experimental research was used. From March to May, 2023, 3 pediatric patients with HS were admitted to the Department of Burns and Cutaneous Surgery of the First Affiliated Hospital of Air Force Medical University, including 2 females and 1 male, aged one year ten months to two years. The HS tissue resected during the surgery and the remaining full-thickness skin graft, i.e., normal skin tissue after full-thickness skin grafting were collected from the aforementioned pediatric patients for subsequent experiments. Using the aforementioned two types of tissue, the histological structures were observed by hematoxylin-eosin staining, collagen distribution was observed by Masson staining, and the expression of CD248 was observed and measured by immunohistochemical staining. The primary HSFs were isolated from HS tissue using explant culture technique, and the 3 to 5 passages of HSFs were used in subsequent experiments. According to the random number table, HSFs were divided into immunoglobulin G78 (IgG78)-treated group and IgG control group, which were treated with 200 nmol/L human CD248 monoclonal antibody IgG78 and human IgG control antibody for 24 h, respectively. The mRNA expressions of collagen type Ⅰ (Col Ⅰ) and α-smooth muscle actin (α-SMA) in HSFs were measured by real-time fluorescence quantitative reverse transcription polymerase chain reaction, the protein expressions of Col Ⅰ and α-SMA in HSFs were detected by Western blotting, and the intracellular location and protein expressions of Col Ⅰ and α-SMA were detected by immunofluorescence method. The number of samples in each experiment was 3. Data were statistically analyzed with paired sample test and independent sample test. Compared with those in normal skin tissue, the epidermis and dermis in HS tissue were significantly thicker, with massive accumulation and disordered arrangement of collagen in the dermis. The expression of CD248 in HS tissue was significantly upregulated compared with that in normal skin tissue (=5.29, <0.05). At post treatment hour 24, the mRNA expressions of Col Ⅰ and α-SMA of HSFs in IgG78-treated group were 0.39±0.05 and 0.56±0.09, respectively, which were significantly lower than 1.00±0.07 and 1.00±0.08 in IgG control group, respectively (with values of 11.87 and 6.49, respectively, values all <0.05). The protein expressions of Col Ⅰ and α-SMA of HSFs in IgG78-treated group were 0.617±0.011 and 0.67±0.14, respectively, which were significantly lower than 1.259±0.052 and 1.23±0.16 in IgG control group, respectively (with values of 20.92 and 4.52, respectively, values all <0.05). At post treatment hour 24, immunofluorescence staining showed that Col Ⅰ and α-SMA mainly located in the cytoplasm of HSFs in the two groups, and the protein expressions of Col Ⅰ and α-SMA of HSFs in IgG78-treated group were obviously downregulated compared with those in IgG control group. The expression of CD248 is significantly upregulated in human HS. Targeted blockade of CD248 can significantly inhibit the collagen synthesis by HSFs and the transdifferentiation of HSFs into myofibroblasts.
探讨内涎蛋白即CD248在人增生性瘢痕（HS）中的表达及其对增生性瘢痕成纤维细胞（HSF）表型的调控作用。采用实验研究方法。2023年3月至5月，空军军医大学第一附属医院烧伤与皮肤外科收治3例小儿HS患者，其中女性2例，男性1例，年龄1岁10个月至2岁。手术切除的HS组织及剩余的全厚皮片即全厚皮片移植后的正常皮肤组织取自上述小儿患者用于后续实验。利用上述两种组织，通过苏木精-伊红染色观察组织结构，通过Masson染色观察胶原分布，通过免疫组化染色观察并检测CD248的表达。采用组织块培养技术从HS组织中分离原代HSF，后续实验采用3至5代的HSF。根据随机数字表，将HSF分为免疫球蛋白G78（IgG78）处理组和IgG对照组，分别用200 nmol/L人CD248单克隆抗体IgG78和人IgG对照抗体处理24小时。通过实时荧光定量逆转录聚合酶链反应检测HSF中Ⅰ型胶原（Col Ⅰ）和α-平滑肌肌动蛋白（α-SMA）的mRNA表达，通过蛋白质印迹法检测HSF中Col Ⅰ和α-SMA的蛋白表达，通过免疫荧光法检测Col Ⅰ和α-SMA的细胞内定位及蛋白表达。每个实验的样本数为3。数据采用配对样本检验和独立样本检验进行统计学分析。与正常皮肤组织相比，HS组织的表皮和真皮明显增厚，真皮中胶原大量堆积且排列紊乱。HS组织中CD248的表达与正常皮肤组织相比明显上调（=5.29，<0.05）。处理后24小时，IgG78处理组HSF中Col Ⅰ和α-SMA的mRNA表达分别为0.39±0.05和0.56±0.09，明显低于IgG对照组的1.00±0.07和1.00±0.08（值分别为11.87和6.49，值均<0.05）。IgG78处理组HSF中Col Ⅰ和α-SMA的蛋白表达分别为0.617±0.011和0.67±0.14，明显低于IgG对照组的1.259±0.052和1.23±0.16（值分别为20.92和4.52，值均<0.05）。处理后24小时，免疫荧光染色显示两组HSF中Col Ⅰ和α-SMA主要位于细胞质中，IgG78处理组HSF中Col Ⅰ和α-SMA的蛋白表达与IgG对照组相比明显下调。CD248在人HS中表达明显上调。靶向阻断CD248可显著抑制HSF的胶原合成及HSF向肌成纤维细胞的转分化。
