Pigment Cell & Melanoma Research最新文献_第7页

Interplay of Light, Melatonin, and Circadian Genes in Skin Pigmentation Regulation 光、褪黑激素和昼夜节律基因在皮肤色素沉着调节中的相互作用。

IF 3.9 3区医学

Pigment Cell & Melanoma Research Pub Date : 2025-01-18 DOI: 10.1111/pcmr.13220

Gabriel E. Bertolesi, Nilakshi Debnath, Neda Heshami, Ryan Bui, Hadi Zadeh-Haghighi, Christoph Simon, Sarah McFarlane

{"title":"Interplay of Light, Melatonin, and Circadian Genes in Skin Pigmentation Regulation","authors":"Gabriel E. Bertolesi, Nilakshi Debnath, Neda Heshami, Ryan Bui, Hadi Zadeh-Haghighi, Christoph Simon, Sarah McFarlane","doi":"10.1111/pcmr.13220","DOIUrl":"10.1111/pcmr.13220","url":null,"abstract":"<p>Circadian regulation of skin pigmentation is essential for thermoregulation, ultraviolet (UV) protection, and synchronization of skin cell renewal. This regulation involves both cell-autonomous photic responses and non-cell-autonomous hormonal control, particularly through melatonin produced in a light-sensitive manner. Photosensitive opsins, cryptochromes, and melatonin regulate circadian rhythms in skin pigment cells. We studied light/dark cycles and melatonin coordination in melanin synthesis and cell proliferation of <i>Xenopus laevis</i> melanophores. In vivo, tadpole pigmentation shows robust circadian regulation mainly hormone-driven, in that isolated melanophores respond strongly to melatonin but only slightly to light. Melanophore proliferation is faster in the dark and slower with melatonin as compared to a 12/12 light/dark cycle. Expression of circadian core genes (<i>clock, bmal1, per1, per2, per3, cry1, cry2</i>, and <i>cry4</i>) in melatonin-treated cells during the light phase mimics dark phase expression. Overexpression of individual Crys did not affect melanization or cell proliferation, likely due to their cooperative actions. Melanin synthesis was inhibited by circadian cycle deregulation through (a) pharmacological inhibition of Cry1 and Cry2 degradation with KL001, (b) continuous light or dark conditions, and (c) melatonin treatment. Our findings suggest that circadian cycle regulation, rather than proliferative capacity, alters melanization of melanophores.</p>","PeriodicalId":219,"journal":{"name":"Pigment Cell & Melanoma Research","volume":"38 1","pages":""},"PeriodicalIF":3.9,"publicationDate":"2025-01-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11742648/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142997133","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Beta-Blockers and Cutaneous Melanoma Outcomes: A Systematic Review and Random-Effects Meta-Analysis 受体阻滞剂和皮肤黑色素瘤的预后：一项系统评价和随机效应荟萃分析。

IF 3.9 3区医学

Pigment Cell & Melanoma Research Pub Date : 2025-01-13 DOI: 10.1111/pcmr.13225

Nicholas M. Muller, Samuel X. Tan, Nisal Vipulaguna, Chenhao Zhou, Maria Celia B. Hughes, H. Peter Soyer, Lena von Schuckmann, Kiarash Khosrotehrani

引用次数: 0

The Incorporation of Melanosomes by Senescent Keratinocytes Causes the Accumulation of Melanin due to Decreased Energy Metabolism 衰老的角质形成细胞合并黑色素小体导致能量代谢减少导致黑色素积累

IF 3.9 3区医学

Pigment Cell & Melanoma Research Pub Date : 2025-01-08 DOI: 10.1111/pcmr.13219

Hiroko Yamazaki, Hideya Ando

{"title":"The Incorporation of Melanosomes by Senescent Keratinocytes Causes the Accumulation of Melanin due to Decreased Energy Metabolism","authors":"Hiroko Yamazaki, Hideya Ando","doi":"10.1111/pcmr.13219","DOIUrl":"https://doi.org/10.1111/pcmr.13219","url":null,"abstract":"<div>\u0000 \u0000 <p>In solar lentigo, a typical age-related pigmentary disorder of the skin, abundant melanin is deposited in the basal layer of keratinocytes and not spontaneously eliminated. The reason for the prolonged melanin accumulation in keratinocytes is not fully understood. Therefore, we focused on the energy metabolism of keratinocytes that incorporate melanosomes, specialized organelles where melanin pigment is synthesized, and investigated the mechanism of melanin accumulation in keratinocytes. Energy metabolism in keratinocytes after the addition of melanosomes was assessed by measuring ATP levels, lactate production, and oxygen consumption rate. Energy limitations after melanosome addition were evaluated by microscopy. Cells with incorporated melanosomes were stained for senescence and proliferation markers. The results showed that keratinocytes upregulated their energy metabolism after melanosome incorporation and energy limitations increased the amount of melanin per cell. Keratinocytes positive for senescence-associated β-galactosidase, a cellular senescence marker, accumulated large amounts of melanin, while keratinocytes positive for 5-ethynyl-2′-deoxyuridine, a proliferation marker, contained little melanin. These findings indicate that senescent keratinocytes tend to accumulate melanin, which may be due to their impaired energy metabolism and thus inability to activate energy metabolism after melanosome incorporation. Our results suggest that melanosome incorporation by senescent keratinocytes causes the persistent melanin deposition in solar lentigo.</p>\u0000 </div>","PeriodicalId":219,"journal":{"name":"Pigment Cell & Melanoma Research","volume":"38 1","pages":""},"PeriodicalIF":3.9,"publicationDate":"2025-01-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143113156","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Association Between Melanoma Metastasis and Metabolic Syndrome: A Cross-Sectional Study in a Chinese Population 黑色素瘤转移与代谢综合征之间的关系：中国人群的横断面研究。

IF 3.9 3区医学

Pigment Cell & Melanoma Research Pub Date : 2024-12-10 DOI: 10.1111/pcmr.13203

Shanshan Sha, Shuomin Sun, Liyun Dong, Haoran Wei, Weiyu Chen, Enzhu Dong, Lu Li, Jiajia Lan, Jun Li, Liu Yang, Yong Chen, Juan Tao

{"title":"Association Between Melanoma Metastasis and Metabolic Syndrome: A Cross-Sectional Study in a Chinese Population","authors":"Shanshan Sha, Shuomin Sun, Liyun Dong, Haoran Wei, Weiyu Chen, Enzhu Dong, Lu Li, Jiajia Lan, Jun Li, Liu Yang, Yong Chen, Juan Tao","doi":"10.1111/pcmr.13203","DOIUrl":"10.1111/pcmr.13203","url":null,"abstract":"<div>\u0000 \u0000 <p>Metabolic syndrome (MetS) remains a significant global public health concern. However, the relationship between MetS, its individual components and melanoma metastasis remains unexplored. We analysed the clinical data of 258 Chinese melanoma patients who had not undergo systemic therapy. Binary logistic regression, adjusted for sex and age, was employed to evaluate the connection between MetS and its components and melanoma metastasis. Of the 258 melanoma patients, 92 met the MetS criteria upon diagnosis. No direct association between MetS and melanoma metastasis was identified. However, specific components of MetS, namely low HDL-cholesterol levels (OR = 2.85, 95% CI:1.50–5.41, <i>p</i> < 0.05) and dysglycaemia (OR = 4.23, 95% CI:1.80–8.96, <i>p</i> < 0.05), were associated with melanoma metastasis. In subgroup analysis, hypertriglyceridemia correlated with melanoma metastasis in non-elderly patients (< 65 years) (OR = 2.69, 95% CI: 1.14–6.33, <i>p</i> < 0.05). Central obesity and hypertension showed no association. A dose–response analysis further indicated that melanoma metastasis risk escalated with increasing fasting blood glucose and blood triglyceride concentrations, and with decreasing blood HDL concentration. Our results suggest that monitoring and managing individual components of the MetS, particularly HDL-cholesterol levels, fasting glucose and triglyceride levels, may have potential prognostic benefits for melanoma in the Chinese population.</p>\u0000 </div>","PeriodicalId":219,"journal":{"name":"Pigment Cell & Melanoma Research","volume":"38 1","pages":""},"PeriodicalIF":3.9,"publicationDate":"2024-12-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142798816","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Curation of OCA2 Variants of Uncertain Significance From Chinese Oculocutaneous Albinism Patients Based on Multiplex Assays 基于多重检测的中国皮肤白化病患者OCA2不确定变异的处理。

IF 3.9 3区医学

Pigment Cell & Melanoma Research Pub Date : 2024-12-05 DOI: 10.1111/pcmr.13212

Qingsong Yang, Yizhen Wang, Zengge Wang, Shushu Lv, Zhenhua Hao, Aihua Wei, Wei Li

{"title":"Curation of OCA2 Variants of Uncertain Significance From Chinese Oculocutaneous Albinism Patients Based on Multiplex Assays","authors":"Qingsong Yang, Yizhen Wang, Zengge Wang, Shushu Lv, Zhenhua Hao, Aihua Wei, Wei Li","doi":"10.1111/pcmr.13212","DOIUrl":"10.1111/pcmr.13212","url":null,"abstract":"<div>\u0000 \u0000 <p>Oculocutaneous albinism type 2 (OCA-2, OMIM: 203200) is associated with variants in the <i>OCA2</i> gene. In this study, we aimed to re-classify variants of uncertain significance (VUS) in OCA2 by evaluating subcellular localization and channel activity through multiplex assays of variant effect (MAVEs). Following the ClinGen guidelines for PS3 evidence, we selected 13 <i>OCA2</i> variants from ClinVar (6 benign/likely benign [B/LB] and 7 pathogenic/likely pathogenic [P/LP]) for OddsPath analysis. The P/LP variants exhibited abnormal functions, while the B/LB variants demonstrated normal functions, supporting the application of “PS3_moderate” evidence for VUS re-classification. In our functional evaluation of 30 VUS identified in 38 individuals with suspected OCA-2 by trio whole-exome sequencing, we observed 6 VUS with abnormal localization and 11 with abnormal channel activity. Based on PS3_moderate evidence, 8 VUS were re-classified as LP, while 22 remained VUS. Consequently, 7 out of 38 previously undiagnosed patients received a molecular diagnosis of OCA-2. These MAVEs offer a robust approach for curating <i>OCA2</i> VUS, enhancing diagnostic accuracy, and informing genetic counseling. Additionally, this variant cohort is a valuable resource for public databases.</p>\u0000 </div>","PeriodicalId":219,"journal":{"name":"Pigment Cell & Melanoma Research","volume":"38 1","pages":""},"PeriodicalIF":3.9,"publicationDate":"2024-12-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142783580","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

High-Risk Melanoma: Stage Before Operating 高危黑色素瘤：手术前阶段。

IF 3.9 3区医学

Pigment Cell & Melanoma Research Pub Date : 2024-12-03 DOI: 10.1111/pcmr.13213

Stanislau Makaranka, Zon Oo, Jonathan Pollock

引用次数: 0

Mouse Tail-Skin Dissociation and Preparation of Live Single-Cell Suspension for Downstream Analysis of Melanocytes 小鼠尾皮解离及单细胞活悬液制备用于黑素细胞下游分析。

IF 3.9 3区医学

Pigment Cell & Melanoma Research Pub Date : 2024-12-03 DOI: 10.1111/pcmr.13216

Vipin Shankar Chelakkot, Kiara Thomas, Leen Hussein, Todd Romigh, Ying Ni, Joshua Arbesman

{"title":"Mouse Tail-Skin Dissociation and Preparation of Live Single-Cell Suspension for Downstream Analysis of Melanocytes","authors":"Vipin Shankar Chelakkot, Kiara Thomas, Leen Hussein, Todd Romigh, Ying Ni, Joshua Arbesman","doi":"10.1111/pcmr.13216","DOIUrl":"10.1111/pcmr.13216","url":null,"abstract":"<div>\u0000 \u0000 <p>Isolating high-quality viable single cells from mouse tail skin, a well-established model for studying skin cells and melanoma pathogenesis, is challenging due to the presence of dense connective tissue and hair follicles. Single-cell RNA sequencing (scRNA-seq) is a powerful tool for studying skin cell heterogeneity. However, the lack of a robust protocol for the efficient generation of highly viable single-cell suspension from mouse tail skin has limited its application for studying melanocyte-interacting cells and characterizing the melanocyte niche. We developed a robust protocol for generating highly viable single-cell suspensions from mouse tail skin, facilitating single-cell transcriptomic profiling of keratinocytes, melanocytes, and fibroblasts. We demonstrate the successful isolation of melanocytes and other melanocyte-interacting cells using our protocol and a proof-of-concept scRNA-seq study for interrogating the melanocyte niche. Our protocol employs a two-stage enzyme dissociation step, followed by debris removal and subsequent live cell enrichment, to obtain a single-cell suspension with high cell viability. This straightforward protocol enables the isolation of viable single cells from mouse tail skin for downstream scRNA-seq studies. Further, this approach allows comprehensive analysis of the melanocyte niche and melanocyte-interacting cells, potentially aiding in identifying the melanoma cell of origin.</p>\u0000 </div>","PeriodicalId":219,"journal":{"name":"Pigment Cell & Melanoma Research","volume":"38 1","pages":""},"PeriodicalIF":3.9,"publicationDate":"2024-12-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142764905","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A Ball Python Colour Morph Implicates MC1R in Melanophore–Xanthophore Distribution and Pattern Formation 一种球蟒的颜色形态与MC1R在黑素团-黄素团分布和模式形成中的关系。

IF 3.9 3区医学

Pigment Cell & Melanoma Research Pub Date : 2024-11-28 DOI: 10.1111/pcmr.13215

Alan Garcia-Elfring, Heather L. Roffey, Jaren M. Abergas, Jurgen Wuyts, Andrew P. Hendry, Athanasia C. Tzika, Rowan D. H. Barrett

{"title":"A Ball Python Colour Morph Implicates MC1R in Melanophore–Xanthophore Distribution and Pattern Formation","authors":"Alan Garcia-Elfring, Heather L. Roffey, Jaren M. Abergas, Jurgen Wuyts, Andrew P. Hendry, Athanasia C. Tzika, Rowan D. H. Barrett","doi":"10.1111/pcmr.13215","DOIUrl":"10.1111/pcmr.13215","url":null,"abstract":"<div>\u0000 \u0000 <p>Reptiles showcase an extensive array of skin colours and patterns, yet little is known about the genetics of reptile colouration. Here, we investigate the genetic basis of the Clown colour morph found in captive-bred ball pythons (<i>Python regius</i>) to study skin pigmentation and patterning in snakes. We obtained samples by crowdsourcing shed skin from commercial breeders and hobbyists. We applied a case–control design, whole-genome pool sequencing, variant annotation, histological analyses, and electron microscopy imaging. We identified a missense mutation in a transmembrane region of the <i>melanocortin-1 receptor</i> (<i>MC1R</i>) associated with the Clown phenotype. In classic avian and mammalian model species, MC1R is known for controlling the type and amount of melanin produced. In contrast, our results suggest that MC1R signalling might play a key role in pattern formation in ball pythons, affecting xanthophore–melanophore distribution. This work highlights the varied functions of MC1R across different vertebrate lineages and promotes a novel model system to study reptile colouration.</p>\u0000 </div>","PeriodicalId":219,"journal":{"name":"Pigment Cell & Melanoma Research","volume":"38 1","pages":""},"PeriodicalIF":3.9,"publicationDate":"2024-11-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142749525","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Novel Variants in Medium and Low Penetrance Predisposing Genes in a Hungarian Malignant Melanoma Cohort With Increased Risk 匈牙利恶性黑色素瘤队列中中外显率和低外显率易感基因的新变异与风险增加。

IF 3.9 3区医学

Pigment Cell & Melanoma Research Pub Date : 2024-11-28 DOI: 10.1111/pcmr.13214

Barbara Anna Bokor, Aliasgari Abdolreza, Flóra Kaptás, Margit Pál, Zita Battyani, Márta Széll, Nikoletta Nagy

{"title":"Novel Variants in Medium and Low Penetrance Predisposing Genes in a Hungarian Malignant Melanoma Cohort With Increased Risk","authors":"Barbara Anna Bokor, Aliasgari Abdolreza, Flóra Kaptás, Margit Pál, Zita Battyani, Márta Széll, Nikoletta Nagy","doi":"10.1111/pcmr.13214","DOIUrl":"10.1111/pcmr.13214","url":null,"abstract":"<div>\u0000 \u0000 <p>Both germline and somatic variants contribute to the genetic background and pathogenesis of melanoma. Germline variants include the presence of rare pathogenic or likely pathogenic variants of high, medium, and low penetrance melanoma-predisposing genes. Rare variants of high penetrance melanoma-predisposing genes are associated with melanoma development, whereas the medium and low penetrance predisposing genes can significantly increase melanoma risk. In this study, we clarified the germline genetic background of a Hungarian melanoma cohort (<i>n</i> = 17). Using a gene panel of 30 melanoma-predisposing genes, germline genetic variants were identified in 10 of the 17 patients (58.82%). A novel, likely pathogenic, missense variant (p.Y143C) in a medium penetrance melanoma-predisposing gene, <i>melanocortin 1 receptor gene</i> (<i>MC1R</i>), and two novel, likely pathogenic nonsense variants in low penetrance genes, p.Q218Ter in caspase 8 (<i>CASP8</i>) and p.Q40Ter in the fat mass- and obesity-associated (<i>FTO</i>) gene were detected. This study highlights the importance of elucidating the germline genetic background of melanoma, which may improve prediction of individual risk and the risk of family members and to optimize preventive, screening, and therapeutic measures for each patient and melanoma-prone families.</p>\u0000 </div>","PeriodicalId":219,"journal":{"name":"Pigment Cell & Melanoma Research","volume":"38 1","pages":""},"PeriodicalIF":3.9,"publicationDate":"2024-11-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142749534","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Exploring the Common Mutational Landscape in Cutaneous Melanoma and Pancreatic Cancer 探讨皮肤黑色素瘤和胰腺癌的常见突变景观。

IF 3.9 3区医学

Pigment Cell & Melanoma Research Pub Date : 2024-11-28 DOI: 10.1111/pcmr.13210

Elisabetta Broseghini, Federico Venturi, Giulia Veronesi, Biagio Scotti, Marina Migliori, Desy Marini, Claudio Ricci, Riccardo Casadei, Manuela Ferracin, Emi Dika

{"title":"Exploring the Common Mutational Landscape in Cutaneous Melanoma and Pancreatic Cancer","authors":"Elisabetta Broseghini, Federico Venturi, Giulia Veronesi, Biagio Scotti, Marina Migliori, Desy Marini, Claudio Ricci, Riccardo Casadei, Manuela Ferracin, Emi Dika","doi":"10.1111/pcmr.13210","DOIUrl":"10.1111/pcmr.13210","url":null,"abstract":"<p>Cutaneous melanoma (CM) and pancreatic cancer are aggressive tumors whose incidences are rapidly increasing in the last years. This review aims to provide a complete and update description about mutational landscape in CM and pancreatic cancer, focusing on similarities of these two apparently so different tumors in terms of site, type of cell involved, and embryonic origin. The familial forms of CM and pancreatic cancers are often characterized by a common mutated gene, namely <i>CDKN2A</i>. In fact, a germline mutation in <i>CDKN2A</i> gene can be responsible for the development of the familial atypical multiple mole and melanoma syndrome (FAMMM), which is characterized by melanomas and pancreatic cancer development. Sporadic melanoma and pancreatic cancer showed different key-driven genes. The open-access resource cBioPortal has been explored to deepen and investigate the common mutational landscape of these two tumors. We investigated the common mutated genes found in both melanoma and pancreatic cancer with a frequency of at least 5% of tested patients and copy number alterations with a frequency of at least of 3%. Data showed that 18 mutated genes and 3 copy number alterations are present in both melanoma and pancreatic cancers types. Since we found two patients that developed both melanoma and pancreatic cancer, we compared mutation landscape between the two tumors and identified a pathogenic variant in <i>BRCA2</i> gene. This review gives valuable insights into the genetic underpinnings of melanoma and pancreatic cancer, urging the continued exploration and research of new genetic biomarkers able to identify patients and families at high risk of developing both cancers and to address to screening and to an effective clinical management of the patient.</p>","PeriodicalId":219,"journal":{"name":"Pigment Cell & Melanoma Research","volume":"38 1","pages":""},"PeriodicalIF":3.9,"publicationDate":"2024-11-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11681848/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142749529","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0