Abstract: Su Mengyun, Lei Tiechi, Yi Wenjuan, Miao Fang, Jiang Shan, Xu Shizheng Department of Dermatology, Renmin Hospital of Wuhan University, Wuhan 430060, China Corresponding author: Lei Tiechi, Email: tchlei@whu.edu.cn 【Abstarct】 Objective To determine the of cathepsin L2（CTSL2）and evaluate its activity in skin lesions of seborrheic keratosis（SK）, to observe the ultrastructural changes of melanosomes in the skin lesions of SK, and to estimate the effect of CTSL2 on the degradation of melanosomes. Methods Twenty patients with SK were enrolled from the Department of Dermatology, Renmin Hospital of Wuhan University. The lesional tissue and the perilesional normal skin were biopsied from each patient. Among 15 of the 20 patients, hematoxylin and eosin（HE）staining and Fontana-Masson silver staining were performed to observe the distribution of melanin granules, transmission electron microscopy（TEM）was conducted to observe the ultrastructural changes of melanosomes, and immunohistochemical staining was performed to estimate the cellular proliferative activity. RT-PCR and fluorogenic substrate cleavage assay were performed in the other 5 patients to determine the mRNA of CTSL2 and evaluate its activity, respectively. Sucrose density gradient ultracentrifugation was performed to isolate and purify melanosomes from the retinal pigment epithelium （RPE） harvested from a discarded eyeball of a 35-year old male patient with informed consent. The purified melanosomes were incubated with epidermal lysates of SK lesions, and TEM was used to observe the changes in the membrane structure of melanosomes. Statistical analysis was carried out by paired t test, and a P value < 0.05 was considered statistically significant. Results A large number of melanin granules were deposited in SK lesions, while the linear deposition of melanin granules was only seen in the basal layer of the normal skin. TEM showed that the percentage of damaged melanosomes was much higher in the normal skin （49.00% ± 4.00%） than in the SK lesions （24.33% ± 3.06%）（t = 8.49, P < 0.05）. RT-PCR revealed that the mRNA and activity of CTSL2 were both significantly lower in the SK lesions than in the normal skin （mRNA: 0.35 ± 0.09 vs. 0.43 ± 0.08, t = 3.17, P < 0.05; activity: 17.46 ± 0.45 vs. 28.78 ± 0.58, t = 34.29, P < 0.05）. Moreover, TEM also showed that the percentage of damaged melanosome was lower in the SK lesion lysate-treated group （32.33% ± 4.93%） than in the normal skin lysate-treated group （43.00% ± 2.65%, t = 3.30, P < 0.05）. Conclusion Decreased of CTSL2 in the SK lesions can affect the degradation of melanosomes by keratinocytes. However, whether CTSL2 directly takes part in the pathogenesis of SK or not is still needed to be further confirmed.

Key words: Keratosis, seborrheic, Melanosomes, Cathepsin L