Abstract: 【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.

Key words: Keloid, Fibroblasts, Myofibroblasts, Papillary fibroblast, Reticular fibroblast, CD90, α-SMA