人真皮网状层成纤维细胞在瘢痕疙瘩皮损组织中的表达与分布

【摘要】 目的 探讨人真皮乳头层成纤维细胞（Fp）、网状层成纤维细胞（Fr）和肌成纤维细胞（MFB）在瘢痕疙瘩皮损组织中的表达与分布。方法 2019年5 - 12月在武汉大学人民医院皮肤科门诊确诊的15例瘢痕疙瘩患者，男8例，女7例，年龄20 ~ 50岁，取皮损组织，以15例年龄匹配的女性乳房整形术正常皮肤组织为对照。采用双重免疫荧光染色法检测成纤维细胞活化蛋白（FAP）、CD90和α平滑肌肌动蛋白（α-SMA）在瘢痕疙瘩和正常皮肤组织中的分布。从3例正常皮肤和3例瘢痕疙瘩组织中分离成纤维细胞原代培养，采用10 ng/ml 转化生长因子β1（TGF-β1）体外处理两组细胞0 ~ 48 h，观察细胞表型的变化，荧光定量RT-PCR和Western印迹检测FAP、CD90和α-SMA mRNA和蛋白表达。两组间差异比较采用t检验。结果 免疫荧光结果显示，正常皮肤组织中，FAP+/CD90-细胞主要分布在真皮浅层，FAP-/CD90+细胞集中在真皮深层，CD90+细胞几乎不表达α-SMA；瘢痕疙瘩组织深层可见大量FAP+和CD90+细胞，大量CD90+细胞同时表达α-SMA。双重免疫荧光染色显示，正常皮肤成纤维细胞几乎不表达α-SMA，瘢痕疙瘩成纤维细胞表达α-SMA；TGF-β1处理24 h时，正常成纤维细胞和瘢痕疙瘩成纤维细胞α-SMA+细胞荧光强度（21.058 ± 0.709、27.112 ± 0.097）均高于未处理组（11.312 ± 0.636、21.306 ± 0.464），t值为22.430、13.370，P < 0.05。RT-PCR和Western印迹显示，TGF-β1处理48 h时，瘢痕疙瘩成纤维细胞FAP、CD90、α-SMA mRNA相对表达水平（92.610 ± 3.667、1.366 ± 0.105、3.240 ± 0.141）与蛋白表达水平（0.652 ± 0.073、1.046 ± 0.119、0.946 ± 0.117）均高于处理前（均P < 0.05）。结论 瘢痕疙瘩组织真皮深层的CD90+（Fr）细胞异常增生，提示针对真皮深层异常增殖活跃的FAP-/CD90+（Fr）细胞群进行定向干预可能提高瘢痕疙瘩治疗疗效。

Expression and distribution of human dermal reticular fibroblasts in keloid tissues

【Abstract】 Objective To investigate the expression and distribution of human dermal papillary fibroblasts （Fp）, reticular fibroblasts （Fr）, and myofibroblasts （MFB）in keloid tissues. Methods Keloid tissues were collected from 15 outpatients （including 8 males and 7 females） aged 20 - 50 years, who were diagnosed in the Department of Dermatology, Renmin Hospital of Wuhan University from May to December 2019. Normal skin tissues were taken from 15 age-matched women who underwent mammoplasty, and served as controls. The distribution of fibroblast activation protein （FAP）, CD90 and alpha-smooth muscle actin （α-SMA） was observed in the keloid tissues and normal skin tissues by dual immunofluorescence staining. Furthermore, fibroblasts were isolated from 3 normal skin and 3 keloid tissue samples, and subjected to primary culture. Subsequently, the fibroblasts were treated with 10 ng/ml transforming growth factor-β1 (TGF-β1) for 48 hours in vitro, during which, changes in fibroblast phenotypes were observed in the 2 groups. Fluorescence-based quantitative RT-PCR and Western blot analysis were performed to determine the mRNA and protein expression of FAP, CD90 and α-SMA. Measurement data were compared between 2 groups by using t test. Results Immunofluorescence staining of the normal skin tissues revealed that FAP+/CD90- fibroblasts were predominantly distributed in the superficial dermis, FAP-/CD90+ fibroblasts in the deep dermis, and CD90+ cells hardly expressed α-SMA; however, a large number of FAP+ fibroblasts and CD90+ fibroblasts were observed in the deep keloid tissues, and many CD90+ fibroblasts also expressed α-SMA. Dual immunofluorescence staining showed that normal tissue-derived fibroblasts hardly expressed α-SMA, and keloid-derived fibroblasts expressed α-SMA. The fluorescence intensity of α-SMA+ cells significantly increased in the normal tissue- and keloid-derived fibroblasts after 24-hour treatment with TGF-β1 （21.058 ± 0.709, 27.112 ± 0.097, respectively） compared with that in the corresponding untreated fibroblasts （11.312 ± 0.636, 21.306 ± 0.464, t = 22.430, 13.370, respectively, both P < 0.05）. RT-PCR and Western blot analysis showed that the mRNA and protein expression of FAP, CD90 and α-SMA significantly increased in the keloid-derived fibroblasts after 48-hour treatment with TGF-β1 （mRNA: 92.610 ± 3.667, 1.366 ± 0.105, 3.240 ± 0.141; protein: 0.652 ± 0.073, 1.046 ± 0.119, 0.946 ± 0.117, respectively） compared with the untreated keloid-derived fibroblasts （all P < 0.05）. Conclusion CD90+ Fr aberrantly proliferated in the deep dermis of keloid tissues, suggesting that directional intervention in aberrantly proliferating FAP-/CD90+ Fr in the deep dermis may promote the efficacy for keloids.