Medical Molecular Morphology最新文献

Transmission electron microscopic analysis of pancreatic ductal adenocarcinoma cell spheres formed in 3D cultures.

IF 1.2 4区医学

Medical Molecular Morphology Pub Date : 2025-04-04 DOI: 10.1007/s00795-025-00435-1

Yuuki Shichi, Masakazu Fujiwara, Fujiya Gomi, Keisuke Nonaka, Fumio Hasegawa, Seiichi Shinji, Hirofumi Rokutan, Tomio Arai, Kimimasa Takahashi, Toshiyuki Ishiwata

{"title":"Transmission electron microscopic analysis of pancreatic ductal adenocarcinoma cell spheres formed in 3D cultures.","authors":"Yuuki Shichi, Masakazu Fujiwara, Fujiya Gomi, Keisuke Nonaka, Fumio Hasegawa, Seiichi Shinji, Hirofumi Rokutan, Tomio Arai, Kimimasa Takahashi, Toshiyuki Ishiwata","doi":"10.1007/s00795-025-00435-1","DOIUrl":"https://doi.org/10.1007/s00795-025-00435-1","url":null,"abstract":"Pancreatic ductal adenocarcinoma (PDAC) cell lines are classified into two types: epithelial and mesenchymal protein-expressing. Using scanning electron microscopy, we reported that these two groups differ in terms of morphology when they formed tumor spheres under three-dimensional (3D) culturing. In this study, we used transmission electron microscopy (TEM) to examine the intracellular microstructures of five epithelial and three mesenchymal PDAC cell lines in 3D culture, and compared them to the morphologies of the same cell types in two-dimensional (2D) cultures. Microvilli were present in all PDAC cells cultured in 2D and 3D, and were well developed in epithelial PDAC cells. Desmosome-like structures were only observed in epithelial PDAC cells, and were more common in 3D cultures. Secretory granules were observed in epithelial PDAC and mesenchymal PANC-1 cells, and were more common in 3D cultures. Intracytoplasmic lumina were only observed in epithelial PK-59 and T3M-4 cells cultured in 3D. Abundant filamentous aggregates were observed in 2D-cultured T3M-4 and MIA PaCa-2 cells. By contrast, entosis was observed in 3D-cultured PK-59, PK-1, and KP4 cells. Microstructural differences enhanced by 3D culturing revealed significant phenotypic diversity among PDAC cells, and may provide key insights into curing intractable pancreatic cancer.","PeriodicalId":18338,"journal":{"name":"Medical Molecular Morphology","volume":" ","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143780173","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"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

Aging potentially reduces CD169 expression in sinus macrophages of pelvic lymph nodes.

IF 1.2 4区医学

Medical Molecular Morphology Pub Date : 2025-03-24 DOI: 10.1007/s00795-025-00433-3

Yuki Ibe, Yojiro Ozaki, Toshiki Anami, Hiromu Yano, Yukio Fujiwara, Hidekazu Nishizawa, Ryoma Kurahashi, Takanobu Motoshima, Yoji Murakami, Junji Yatsuda, Yoshihiro Komohara, Tomomi Kamba

{"title":"Aging potentially reduces CD169 expression in sinus macrophages of pelvic lymph nodes.","authors":"Yuki Ibe, Yojiro Ozaki, Toshiki Anami, Hiromu Yano, Yukio Fujiwara, Hidekazu Nishizawa, Ryoma Kurahashi, Takanobu Motoshima, Yoji Murakami, Junji Yatsuda, Yoshihiro Komohara, Tomomi Kamba","doi":"10.1007/s00795-025-00433-3","DOIUrl":"https://doi.org/10.1007/s00795-025-00433-3","url":null,"abstract":"The number of patients with prostate cancer has been increasing around the world. Although anticancer immunotherapy targeting the immune checkpoint molecules has been approved for many types of cancer, no significant anti-cancer effects have been observed in patients with prostate cancer. Lymph node sinus macrophages (LSMs) are known to work as antigen-presenting cells, which are critical for anticancer immune responses. Previous studies have suggested that CD169 expression in LSMs affects anticancer immune responses in several cancers, including prostate cancer. In the present study, we aimed to examine the correlation between the tumor immune microenvironment and activation status of LSMs in patients with prostate cancer. Forty-two cases of high-risk localized prostate cancer treated using robot-assisted laparoscopic radical prostatectomy and lymph node dissection between 2017 and 2021 were enrolled. CD169 expression in LSMs was examined by immunohistochemistry. The results indicated that CD169 expression in LSMs was significantly decreased in older (≥ 75 years) compared with younger patients. However, no significant correlation was found between CD169 expression and any other clinicopathological factors. In addition, CD3- and CD8-postitive lymphocytes in primary cancer tissues were evaluated in the same cases, and their correlations with CD169 expression in LSMs were tested. Although these lymphocytes tended to be higher in CD169high than in CD169low cases, the difference was not statistically significant. In conclusion, we found that CD169 expression was upregulated in older patients and tended to be related to T cell infiltration in cancer tissues. Therefore, the downregulation of CD169 in LSMs might be involved in the reduced anticancer immune response in prostate cancer.","PeriodicalId":18338,"journal":{"name":"Medical Molecular Morphology","volume":" ","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-03-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143700596","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"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

The cross-talk between NRF2 and apoptosis in cancer.

IF 1.2 4区医学

Medical Molecular Morphology Pub Date : 2025-03-24 DOI: 10.1007/s00795-025-00434-2

Elmira Aboutalebi Vand Beilankouhi, Bahareh Yousefi, Niloofar Sadat Hadian, Reza Safaralizadeh, Mohammad Valilo

{"title":"The cross-talk between NRF2 and apoptosis in cancer.","authors":"Elmira Aboutalebi Vand Beilankouhi, Bahareh Yousefi, Niloofar Sadat Hadian, Reza Safaralizadeh, Mohammad Valilo","doi":"10.1007/s00795-025-00434-2","DOIUrl":"https://doi.org/10.1007/s00795-025-00434-2","url":null,"abstract":"Cancer is one of the common diseases that affects people in the society, the prevalence of which has decreased somewhat in recent years. Various genetic and environmental factors play a role in the development and progression of cancer. NRF2 is a transcriptional regulator that controls the expression of antioxidant response element-related genes. It plays an important role in regulating the physiological and pathophysiological consequences of oxidant exposure. NRF2 is also responsible for regulating the expression of various cellular protective genes. NRF2 activity is regulated at multiple levels including protein stability, transcription, and post-transcription. The Keap1-Cul3-Rbx1 axis is the most prominent regulator of NRF2 activity. Apoptosis is a type of programmed cell death that is initiated by two intrinsic and extrinsic pathways. Caspases play a major role in this cell death pathway. Apoptosis pathway is related to many cells signaling pathways that are interconnected. Disruption in one pathway affects the other pathway. One of these signaling pathways is the NRF2 pathway, which is associated with apoptosis, which are interconnected and play an important role in disease prevention or progression. Therefore, in this study, we decided to investigate the relationship between NRF2 and apoptosis in cancer.","PeriodicalId":18338,"journal":{"name":"Medical Molecular Morphology","volume":" ","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-03-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143700600","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"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

Expression of Bax and Bcl-2 in the placenta from pre-term, term, and post-term deliveries: immunohistochemical analysis.

IF 1.2 4区医学

Medical Molecular Morphology Pub Date : 2025-03-19 DOI: 10.1007/s00795-025-00429-z

Xvni Tang, Liyan Ye, Ting Yan, Xiujuan Zheng, Lichao Yuan

{"title":"Expression of Bax and Bcl-2 in the placenta from pre-term, term, and post-term deliveries: immunohistochemical analysis.","authors":"Xvni Tang, Liyan Ye, Ting Yan, Xiujuan Zheng, Lichao Yuan","doi":"10.1007/s00795-025-00429-z","DOIUrl":"https://doi.org/10.1007/s00795-025-00429-z","url":null,"abstract":"The placenta is the most critical organ during pregnancy and its developmental status directly impacts fetal health. Placental dysfunction is associated with various pregnancy complications, including preterm birth, fetal growth restriction, and premature rupture of membranes (PROM). This study aimed to elucidate the expression changes of Bax and Bcl-2 and their association with pregnancy-related complications, providing new insights into placental dysfunction during pregnancy and offering a theoretical foundation for clinical diagnosis and prevention. The placental samples from 118 late pregnant women were retrospectively analyzed. They were assigned into pre-term, term, post-term, PROM, and non-PROM groups based on gestational age and the occurrence of PROM. Immunohistochemistry (IHC) staining was performed to gauge the Bax and Bcl-2 expressions in placenta. Receiver operating characteristic (ROC) curve was subsequently conducted to assess their associated with efficacy for PROM. The weight and volume of placentas in the pre-term group were sharply smaller to those in the term and post-term groups, with no apparent fibrosis or calcification observed. The term and post-term groups exhibited marked elevated Bax expression in the parturient group in contrast to the non-parturient group (P < 0.05), while there existed no substantial significance in Bcl-2 expression. The area under the curve (AUC) for Bax and Bcl-2 was 0.975 and 0.596, respectively, with a combined associated with value of 0.978, higher to single predictions. Bax and Bcl-2 expressions in late pregnancy placentas were associated with the onset of parturient status and PROM and their combined prediction exhibited accurate PROM predicted. These findings offered a new perspective for understanding the physiological and pathological changes of the placenta during pregnancy.","PeriodicalId":18338,"journal":{"name":"Medical Molecular Morphology","volume":" ","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-03-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143657627","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"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

Loss of miR-424 and miR-503 promotes decidualization of human endometrial stromal cells by increasing SCARA5 expression. miR-424和miR-503的缺失通过增加SCARA5的表达促进人子宫内膜基质细胞的蜕膜化。

IF 1.2 4区医学

Medical Molecular Morphology Pub Date : 2025-03-14 DOI: 10.1007/s00795-025-00431-5

Tetsu Yamaguchi, Masashi Takamura, Hideno Tochigi, Yumi Mizuno, Yosuke Mizuno, Tomomi Sato, Shunsuke Tamaru, Kazuya Kusama, Kazuhiro Tamura, Yoshimasa Kamei, Takeshi Kajihara

{"title":"Loss of miR-424 and miR-503 promotes decidualization of human endometrial stromal cells by increasing SCARA5 expression.","authors":"Tetsu Yamaguchi, Masashi Takamura, Hideno Tochigi, Yumi Mizuno, Yosuke Mizuno, Tomomi Sato, Shunsuke Tamaru, Kazuya Kusama, Kazuhiro Tamura, Yoshimasa Kamei, Takeshi Kajihara","doi":"10.1007/s00795-025-00431-5","DOIUrl":"https://doi.org/10.1007/s00795-025-00431-5","url":null,"abstract":"This study aims to investigate the function of miR-424 and miR-503, identified as putative regulatory miRNAs of FOXO1, a key factor for decidualization. The expression of both miR-424 and miR-503 in human endometrial stromal cells (HESCs) were measured before and after decidualization. Then, HESCs were transfected with both miR-424 and miR-503 before decidualization. Quantitative reverse transcription PCR, actin staining analysis, migration assay, fluorescence immunostaining, and luciferase assay were performed. MiR-424 and miR-503 expression was decreased after decidualization. Overexpression of both miR-424 and miR-503 inhibited major decidual maker genes, including FOXO1, PRL, IGFBP1, WNT4, and SCARA5, and altered F-actin's subcellular distribution from the periphery to all over the cytoplasm, concomitantly increasing cell mobility. Moreover, immunohistochemical analysis revealed overexpression of both miRNAs resulted in FOXO1 protein accumulation in the cytoplasm. Knocking down FOXO1 decreased SCARA5 expression, revealing SCARA5 is a downstream target of FOXO1. In addition, a luciferase reporter assay confirmed that the 3'-untranslated region of FOXO1 mRNA is targeted by miR-424. These results suggest that both miRNAs may play an important role in endometrial decidualization by regulating transcriptional activity of FOXO1, which alters decidualization-related gene expression such as SCARA5.Abstract: Journal standard instruction requires an unstructured abstract; hence structured abstract changed to unstructured.Thank you for the correction. I approve this change.","PeriodicalId":18338,"journal":{"name":"Medical Molecular Morphology","volume":" ","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-03-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143630601","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"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

Large number of CD68⁺/CD163⁺ Hofbauer cells and characteristic vascular structural alterations in the placental villi of cases with placenta accreta spectrum.

IF 1.2 4区医学

Medical Molecular Morphology Pub Date : 2025-03-13 DOI: 10.1007/s00795-025-00432-4

Hazuki Kashiwagi, Kengo Shigehara, Terufumi Kubo, Yoshihiko Hirohashi, Tasuku Mariya, Kazuhiko Matsuo, Tomoyuki Minowa, Shin-Ichi Ishioka, Kenji Murata, Takayuki Kanaseki, Tomohide Tsukahara, Tadashi Hasegawa, Tsuyoshi Saito, Toshihiko Torigoe

{"title":"Large number of CD68+/CD163+ Hofbauer cells and characteristic vascular structural alterations in the placental villi of cases with placenta accreta spectrum.","authors":"Hazuki Kashiwagi, Kengo Shigehara, Terufumi Kubo, Yoshihiko Hirohashi, Tasuku Mariya, Kazuhiko Matsuo, Tomoyuki Minowa, Shin-Ichi Ishioka, Kenji Murata, Takayuki Kanaseki, Tomohide Tsukahara, Tadashi Hasegawa, Tsuyoshi Saito, Toshihiko Torigoe","doi":"10.1007/s00795-025-00432-4","DOIUrl":"https://doi.org/10.1007/s00795-025-00432-4","url":null,"abstract":"Placenta accreta spectrum (PAS) is a serious disease leading to complications and maternal death. The objective of the study was to characterize the placental villi and blood vessels of PAS villi histopathologically. We investigated 10 cases of PAS (five cases of placenta increta, two cases of placenta accreta, and three cases of placenta percreta) histologically. Immunohistochemical staining using anti-CD68 or anti-CD163 antibodies was performed to detect and count Hofbauer cells. Immunohistochemical staining with an anti-CD34 antibody was used to detect vascular endothelial cells, and the number and area of vessels were analyzed. The numbers of CD68-positive or CD163-positive Hofbauer cells were larger in PAS cases compared with control cases. The vascular area in villi was smaller in PAS cases compared with control cases. The number of blood vessels in villi was slightly higher in PAS cases than in control cases. The numbers of Hofbauer cells and vessels in villi were larger in PAS cases compared with control cases, whereas the area of vessels in villi was smaller in PAS cases compared with control cases. Although their biological meaning is elusive, these findings provide novel insights into the pathogenesis of PAS, particularly regarding the role of Hofbauer cells in immune-suppressive role and angiogenesis and the alterations in vascular structure and hemodynamics in the chorionic villi.","PeriodicalId":18338,"journal":{"name":"Medical Molecular Morphology","volume":" ","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-03-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143625313","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"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 type of pancreatic cancer cells form cell clusters from a solitary condition in a primary ciliogenesis-dependent manner.

IF 1.2 4区医学

Medical Molecular Morphology Pub Date : 2025-03-12 DOI: 10.1007/s00795-025-00428-0

Kenji Shirakawa, Ryota Nakazato, Tetsuhiro Hara, Kenichiro Uemura, Faryal Ijaz, Shinya Takahashi, Koji Ikegami

{"title":"A type of pancreatic cancer cells form cell clusters from a solitary condition in a primary ciliogenesis-dependent manner.","authors":"Kenji Shirakawa, Ryota Nakazato, Tetsuhiro Hara, Kenichiro Uemura, Faryal Ijaz, Shinya Takahashi, Koji Ikegami","doi":"10.1007/s00795-025-00428-0","DOIUrl":"https://doi.org/10.1007/s00795-025-00428-0","url":null,"abstract":"Primary cilia are hair-like projections that protrude on most of mammalian cells and mediate reception of extracellular signals. Numerous studies have demonstrated that a variety of cancer cells including pancreatic ductal adenocarcinoma (PDAC) fail to form primary cilia. The loss of primary cilia is thought to cause carcinogenesis and progressive cell proliferation. However, the relationship of the primary cilia loss with carcinogenesis and/or cancer malignancy remains arguable. We herein examined whether ciliogenesis was increased in a model of more progressive PDAC and investigated effects of ciliogenesis on growth of PDAC using a pancreatic cancer cell line, PANC-1. The majority of PANC-1 cells in a cell cluster grown from a solitary cell possessed primary cilia. The rate of ciliogenesis was higher in cells grown from low density than in cells grown from high density. Almost all clones passing limiting dilution culture had abilities to grow primary cilia. Compared to the parental PANC-1 cells, clones that proliferated from a solitary cell showed increase in the ciliogenesis rate. Blocking ciliogenesis suppressed cell cluster formation. Our results suggest that pancreatic cancer cells that are more resistant to a solitary condition have abilities of ciliogenesis and form tumor-like cell clusters in a primary cilia-dependent manner.","PeriodicalId":18338,"journal":{"name":"Medical Molecular Morphology","volume":" ","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-03-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143605468","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"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

Androgens modulate endometrial function.

IF 1.2 4区医学

Medical Molecular Morphology Pub Date : 2025-03-10 DOI: 10.1007/s00795-025-00430-6

Ko Yamagata, Yousuke Mizuno, Yumi Mizuno, Shunnsuke Tamaru, Takeshi Kajihara

引用次数: 0

Novel orthotopic patient-derived xenograft model using human pancreatic cancer tissue fragments to recapitulate distant metastasis and cancer-related hypercoagulability.

IF 1.2 4区医学

Medical Molecular Morphology Pub Date : 2025-03-10 DOI: 10.1007/s00795-025-00425-3

Takuma Miura, Arisa Watanabe, Mutsumi Miyake, Sayaka Suga, Makoto Miyoshi, Kumiko Miyashita, Shohei Komatsu, Noriyuki Nishimura, Kazuya Shimizu, Yuichi Hori

{"title":"Novel orthotopic patient-derived xenograft model using human pancreatic cancer tissue fragments to recapitulate distant metastasis and cancer-related hypercoagulability.","authors":"Takuma Miura, Arisa Watanabe, Mutsumi Miyake, Sayaka Suga, Makoto Miyoshi, Kumiko Miyashita, Shohei Komatsu, Noriyuki Nishimura, Kazuya Shimizu, Yuichi Hori","doi":"10.1007/s00795-025-00425-3","DOIUrl":"https://doi.org/10.1007/s00795-025-00425-3","url":null,"abstract":"Pancreatic cancer is a malignant tumor that metastasizes to distant organs, such as the liver and lungs from an early stage. Few animal models can reproduce early metastasis. In addition, no model has been reported that reproduces cancer-related hypercoagulability, which is characteristic of pancreatic ductal adenocarcinoma and other adenocarcinomas. We hypothesized that the reason why the commonly used orthotopic cell xenograft model cannot reproduce the disease is inadequate construction of the cancer microenvironment. We developed an orthotopic tissue fragment xenograft model in which tumor tissue was transplanted into the pancreas of mice while preserving the microenvironment. Briefly, we injected single cancer cells subcutaneously to form a tumor, which was then cut with a scalpel into tumor fragments. A fragment was then sutured and fixed to the surface of the pancreatic tail. In this study, we evaluated the superiority of this model over a conventional orthotopic cell xenograft model. As a result, the novel orthotopic tissue xenograft model reproduced early distant metastasis to the liver and lung, nerve invasion, and cancer-related hypercoagulability of human pancreatic cancer, and showed greater similarity to clinical cases than the control orthotopic cell xenograft model.","PeriodicalId":18338,"journal":{"name":"Medical Molecular Morphology","volume":" ","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-03-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143597272","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"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

Three-dimensional visualization of calcification during scale regeneration in goldfish. 金鱼鳞片再生过程中钙化的三维可视化。

IF 1.2 4区医学

Medical Molecular Morphology Pub Date : 2025-03-06 DOI: 10.1007/s00795-025-00427-1

Hisayuki Funahashi, Yusuke Maruyama, Nobuo Suzuki, Takashi Takaki, Kazuho Honda, Atsuhiko Hattori

引用次数: 0