Expression of endosialin in human hypertrophic scars and its regulation on fibroblast phenotype
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摘要:
目的 探讨内皮唾液酸蛋白即CD248在人增生性瘢痕（HS）中的表达及其对人HS成纤维细胞（HSF）表型的调控作用。 方法 采用实验研究方法。2023年3—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HS患儿，其中女2例、男1例，年龄1岁10个月~2岁。取前述患儿术中切除的HS组织及行全厚皮移植术后剩余的全厚皮片即正常皮肤组织，进行后续实验。取前述2种组织，行苏木精-伊红染色后观察组织结构，行Masson染色后观察组织中胶原分布情况，行免疫组织化学染色后观测组织中CD248表达情况。取HS组织，采用组织块培养法提取原代HSF，取第3~5代HSF进行后续实验。将HSF按随机数字表法分为免疫球蛋白G78（IgG78）处理组与IgG对照组，分别用终物质的量浓度为200 nmol/L的人源性CD248单克隆抗体IgG78与人源性IgG对照抗体处理24 h，采用实时荧光定量反转录PCR法检测细胞中Ⅰ型胶原蛋白和α平滑肌肌动蛋白（α-SMA）的mRNA表达，采用蛋白质印迹法检测细胞中Ⅰ型胶原蛋白和α-SMA的蛋白表达，采用免疫荧光法检测细胞中Ⅰ型胶原蛋白和α-SMA的定位和蛋白表达情况。各实验样本数均为3。对数据行配对样本 t检验和独立样本 t检验。 结果 相较于正常皮肤组织，HS组织中表皮与真皮层均明显增厚，真皮层中胶原大量蓄积且排布紊乱。HS组织中CD248表达量明显高于正常皮肤组织（ t=5.29， P<0.05）。处理24 h，IgG78处理组HSF中Ⅰ型胶原蛋白、α-SMA的mRNA表达量分别为0.39±0.05、0.56±0.09，分别明显低于IgG对照组的1.00±0.07、1.00±0.08（ t值分别为11.87、6.49， P值均<0.05）；IgG78处理组HSF中Ⅰ型胶原蛋白、α-SMA的蛋白表达量分别为0.617±0.011、0.67±0.14，分别明显低于IgG对照组的1.259±0.052、1.23±0.16（ t值分别为20.92、4.52， P值均<0.05）。处理24 h，免疫荧光染色显示，Ⅰ型胶原蛋白、α-SMA主要定位在2组HSF的细胞质中，IgG78处理组HSF中Ⅰ型胶原蛋白、α-SMA的蛋白表达均较IgG对照组明显减少。 结论 CD248在人HS中表达显著增高，靶向阻断CD248能够显著抑制人HSF的胶原合成与向肌Fb的转分化。 Abstract:Objective 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). Methods 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 rd to 5 th 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 t test and independent sample t test. Results 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 ( t=5.29, P<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 t values of 11.87 and 6.49, respectively, P 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 t values of 20.92 and 4.52, respectively, P 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. Conclusions 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. -
Key words:
- Cicatrix /
- Fibroblasts /
- Extracellular matrix /
- Cell transdifferentiation /
- Endosialin /
- Collagen synthesis
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参考文献
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1 人增生性瘢痕（HS）组织与正常皮肤组织结构和胶原分布及CD248的表达情况。1A、1D.分别为正常皮肤组织和HS组织结构情况，图1D的表皮与真皮层均较图1A增厚 苏木精-伊红×15；1B、1E.分别为正常皮肤组织和HS组织中胶原情况，图1E中胶原（蓝色）排列相较图1B紊乱且较为致密 Masson×15；1C、1F.分别为正常皮肤组织和HS组织中CD248表达情况，图1F中CD248（阳性染色为黄色/棕色）表达量显著高于图1C 二氨基联苯胺×15
注：CD248即为内皮唾液酸蛋白
2 蛋白质印迹法检测的2组人增生性瘢痕来源成纤维细胞处理24 h后Ⅰ型胶原蛋白和α-SMA的蛋白表达
注：α-SMA为α平滑肌肌动蛋白；1、2分别指示IgG对照组、IgG78处理组；IgG为免疫球蛋白G，IgG78为人源性内皮唾液酸蛋白即CD248单克隆抗体
3 2组人增生性瘢痕来源成纤维细胞处理24 h后Ⅰ型胶原蛋白和α-SMA定位与蛋白表达 Alexa Fluor 488-Alexa Fluor 594-4',6-二脒基-2-苯基吲哚×60。3A、3B、3C.分别为IgG对照组细胞核染色、Ⅰ型胶原蛋白染色、细胞核与Ⅰ型胶原蛋白染色重叠图片，细胞核完整，Ⅰ型胶原蛋白的蛋白表达较多且主要定位在细胞质中；3D、3E、3F.分别为IgG78处理组细胞核染色、Ⅰ型胶原蛋白染色、细胞核与Ⅰ型胶原蛋白染色重叠图片，细胞核完整，图3E细胞质中Ⅰ型胶原蛋白的蛋白表达较图3B明显减少；3G、3H、3I.分别为IgG对照组细胞核染色、α-SMA染色、细胞核与α-SMA染色重叠图片，细胞核完整，α-SMA蛋白表达较多且主要定位在细胞质中；3J、3K、3L.分别为IgG78处理组细胞核染色、α-SMA染色、细胞核与α-SMA染色重叠图片，细胞核完整，图3K细胞质中α-SMA蛋白表达较图3H明显减少
注：α-SMA为α平滑肌肌动蛋白，IgG为免疫球蛋白G，IgG78为人源性内皮唾液酸蛋白即CD248单克隆抗体；细胞核阳性染色为蓝色，Ⅰ型胶原蛋白阳性染色为绿色，α-SMA阳性染色为红色
表1 2组人增生性瘢痕来源成纤维细胞处理24 h后Ⅰ型胶原蛋白和α-SMA的mRNA与蛋白表达比较（
表1. Comparison of the mRNA and protein expres‑sions of collagen Ⅰ and α-SMA of two groups of human hypertrophic scar-derived fibro‑blasts after 24 h of treatment
组别 样本量 Ⅰ型胶原蛋白 α-SMA mRNA 蛋白 mRNA 蛋白 IgG78处理组 3 0.39±0.05 0.617±0.011 0.56±0.09 0.67±0.14 IgG对照组 3 1.00±0.07 1.259±0.052 1.00±0.08 1.23±0.16 t值 11.87 20.92 6.49 4.52 P值 <0.001 <0.001 0.003 0.011 注：α-SMA为α平滑肌肌动蛋白，IgG为免疫球蛋白G，IgG78为人源性内皮唾液酸蛋白即CD248单克隆抗体 
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