Objective:To establish a complete system for the isolation, purification, identification, and culture of Kaposiform hemangioendothelioma-derived endothelial cells (KHE-ECs), to analyze the phenotype of KHE-ECs, and to explore the possibility of establishing a KHE-EC bank.Methods:A novel digestion solution for KHE tumors (patent number: CN202410500224.2) was formulated using collection fluid, Liberase TM and dispase stock solutions, and was used to process tumor tissues to obtain cells. High-purity KHE-ECs were purified using CD31 + immunomagnetic beads. The EGM-2 complete medium containing 10% fetal bovine serum and 2% penicillin-streptomycin solution was employed for cell culture. To verify the characteristics of KHE-ECs, immunofluorescence assay was conducted to determine the expression of endothelial cell-specific markers CD31 and CD34, KHE disease markers podoplanin (D2-40), prospero-related homeobox 1 (Prox-1), and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), as well as an infantile hemangioma-specific diagnostic marker glucose transporter 1 (GLUT-1). Human umbilical vein endothelial cells (HUVECs) served as controls for the phenotype analysis of KHE-ECs, including cell viability, cytoskeleton, proliferation, migration, invasion, tube formation, and sprouting ability. Results:Primary cells were successfully isolated from KHE tumor tissues, and high-purity KHE-ECs were obtained by using CD31 + immunomagnetic beads. The cells exhibited typical spindle-shaped morphology and an adherent growth pattern. Immunofluorescence assay showed that KHE-ECs expressed CD31, CD34, D2-40, Prox-1, and LYVE1, but did not express GLUT-1. There were significant differences in cell morphology, cell viability, and cytoskeletal structures between KHE-ECs and HUVECs. Additionally, the KHE-EC group showed significantly increased percentages of proliferative cells (29.1% ± 2.5%), numbers of migratory cells (114.3 ± 9.4) and invasive cells (110.0 ± 6.1), tube length (32 121.0 ± 892.0 μm), and number of sprouting cells (25.0 ± 3.6) compared with the HUVEC group (13.0% ± 2.2%, 38.0 ± 3.6, 35.3 ± 2.3, 25 345.0 ± 448.1 μm, 5.0 ± 1.0, respectively, all P ≤ 0.001) . Conclusion:An innovative digestion solution specifically for KHE tumors was formulated for the first time, and high-purity and well-growing KHE-EC strains were successfully isolated and purified by using the novel digestion solution in combination with CD31 + immunomagnetic beads, providing a stable and reliable cell source for subsequent experimental studies on KHE and laying the foundation for establishing a KHE-EC bank.

Objective:To establish a complete system for the isolation, purification, identification, and culture of Kaposiform hemangioendothelioma-derived endothelial cells (KHE-ECs), to analyze the phenotype of KHE-ECs, and to explore the possibility of establishing a KHE-EC bank.Methods:A novel digestion solution for KHE tumors (patent number: CN202410500224.2) was formulated using collection fluid, Liberase TM and dispase stock solutions, and was used to process tumor tissues to obtain cells. High-purity KHE-ECs were purified using CD31 + immunomagnetic beads. The EGM-2 complete medium containing 10% fetal bovine serum and 2% penicillin-streptomycin solution was employed for cell culture. To verify the characteristics of KHE-ECs, immunofluorescence assay was conducted to determine the expression of endothelial cell-specific markers CD31 and CD34, KHE disease markers podoplanin (D2-40), prospero-related homeobox 1 (Prox-1), and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), as well as an infantile hemangioma-specific diagnostic marker glucose transporter 1 (GLUT-1). Human umbilical vein endothelial cells (HUVECs) served as controls for the phenotype analysis of KHE-ECs, including cell viability, cytoskeleton, proliferation, migration, invasion, tube formation, and sprouting ability. Results:Primary cells were successfully isolated from KHE tumor tissues, and high-purity KHE-ECs were obtained by using CD31 + immunomagnetic beads. The cells exhibited typical spindle-shaped morphology and an adherent growth pattern. Immunofluorescence assay showed that KHE-ECs expressed CD31, CD34, D2-40, Prox-1, and LYVE1, but did not express GLUT-1. There were significant differences in cell morphology, cell viability, and cytoskeletal structures between KHE-ECs and HUVECs. Additionally, the KHE-EC group showed significantly increased percentages of proliferative cells (29.1% ± 2.5%), numbers of migratory cells (114.3 ± 9.4) and invasive cells (110.0 ± 6.1), tube length (32 121.0 ± 892.0 μm), and number of sprouting cells (25.0 ± 3.6) compared with the HUVEC group (13.0% ± 2.2%, 38.0 ± 3.6, 35.3 ± 2.3, 25 345.0 ± 448.1 μm, 5.0 ± 1.0, respectively, all P ≤ 0.001) . Conclusion:An innovative digestion solution specifically for KHE tumors was formulated for the first time, and high-purity and well-growing KHE-EC strains were successfully isolated and purified by using the novel digestion solution in combination with CD31 + immunomagnetic beads, providing a stable and reliable cell source for subsequent experimental studies on KHE and laying the foundation for establishing a KHE-EC bank.

Objective:To investigate the optimal timing of oral propranolol treatment for proliferative infantile hemangiomas (IH) .Methods:A bidirectional cohort study was conducted. Infants with proliferative IH receiving oral propranolol treatment were collected from the Department of Pediatric Surgery, West China Hospital, Sichuan University between June 2015 and May 2019, and their general information and IH-related clinical data were analyzed. The primary outcome was the satisfactory regression rate of IH during 6-12 months of continuous oral propranolol treatment; secondary outcomes included the time to achieve satisfactory regression, incidence of adverse reactions, incidence of IH ulceration, and IH recurrence rate. Multivariate logistic regression was performed to identify factors influencing the satisfactory regression of IH after propranolol treatment, and a receiver operating characteristic (ROC) curve was employed to determine the optimal age for initiating propranolol therapy.Results:A total of 122 IH infants were enrolled in the study, including 32 males (26.2%) and 90 females (73.8%), with ages ( M[ Q1, Q3]) of 8.6 [6.3, 12.3] weeks. IH was located on the head and face in 56 cases (45.9%). There were 57 cases (46.7%) of localized IH, 53 (43.4%) of segmental IH, and 86 (70.5%) of mixed-type IH. Ulceration occurred in 17 cases (13.9%). After 6 months of propranolol treatment, 8 patients (6.6%) experienced treatment failure, and 12 (9.8%) experienced relapse within 6 months after discontinuation of propranolol. During 6 months of oral propranolol treatment, 56 infants (45.9%) experienced mild to moderate adverse reactions, with no drug-related deaths observed. Multivariate logistic regression analysis revealed that the age at initiation of propranolol treatment was an independent factor influencing satisfactory regression of IH ( OR = 0.879, 95% CI: 0.808 - 0.957). ROC curve analysis revealed that the optimal age for starting propranolol therapy was 9.9 weeks, with a sensitivity of 75.7% and a specificity of 61.5%. Infants aged ≤ 9.9 weeks (73 cases) had a significantly higher satisfactory regression rate (72.6% [53/73]) compared with those aged > 9.9 weeks (49 cases, 34.7% [17/49]; χ2 = 17.23, P < 0.001) ; the time to achieve satisfactory regression of IH was significantly shorter in the infants aged ≤ 9.9 weeks ( M[ Q1, Q3]: 46.0 [38.5, 48.0] weeks) than in those aged > 9.9 weeks (57.0 [40.0, 73.5] weeks; Z = -2.01, P = 0.045) . Conclusion:For IH infants requiring systemic therapy, initiation of oral propranolol before the age of 10 weeks appeared to improve the satisfactory regression rate of IH.

Objective:To investigate the optimal timing of oral propranolol treatment for proliferative infantile hemangiomas (IH) .Methods:A bidirectional cohort study was conducted. Infants with proliferative IH receiving oral propranolol treatment were collected from the Department of Pediatric Surgery, West China Hospital, Sichuan University between June 2015 and May 2019, and their general information and IH-related clinical data were analyzed. The primary outcome was the satisfactory regression rate of IH during 6-12 months of continuous oral propranolol treatment; secondary outcomes included the time to achieve satisfactory regression, incidence of adverse reactions, incidence of IH ulceration, and IH recurrence rate. Multivariate logistic regression was performed to identify factors influencing the satisfactory regression of IH after propranolol treatment, and a receiver operating characteristic (ROC) curve was employed to determine the optimal age for initiating propranolol therapy.Results:A total of 122 IH infants were enrolled in the study, including 32 males (26.2%) and 90 females (73.8%), with ages ( M[ Q1, Q3]) of 8.6 [6.3, 12.3] weeks. IH was located on the head and face in 56 cases (45.9%). There were 57 cases (46.7%) of localized IH, 53 (43.4%) of segmental IH, and 86 (70.5%) of mixed-type IH. Ulceration occurred in 17 cases (13.9%). After 6 months of propranolol treatment, 8 patients (6.6%) experienced treatment failure, and 12 (9.8%) experienced relapse within 6 months after discontinuation of propranolol. During 6 months of oral propranolol treatment, 56 infants (45.9%) experienced mild to moderate adverse reactions, with no drug-related deaths observed. Multivariate logistic regression analysis revealed that the age at initiation of propranolol treatment was an independent factor influencing satisfactory regression of IH ( OR = 0.879, 95% CI: 0.808 - 0.957). ROC curve analysis revealed that the optimal age for starting propranolol therapy was 9.9 weeks, with a sensitivity of 75.7% and a specificity of 61.5%. Infants aged ≤ 9.9 weeks (73 cases) had a significantly higher satisfactory regression rate (72.6% [53/73]) compared with those aged > 9.9 weeks (49 cases, 34.7% [17/49]; χ2 = 17.23, P < 0.001) ; the time to achieve satisfactory regression of IH was significantly shorter in the infants aged ≤ 9.9 weeks ( M[ Q1, Q3]: 46.0 [38.5, 48.0] weeks) than in those aged > 9.9 weeks (57.0 [40.0, 73.5] weeks; Z = -2.01, P = 0.045) . Conclusion:For IH infants requiring systemic therapy, initiation of oral propranolol before the age of 10 weeks appeared to improve the satisfactory regression rate of IH.

Objective:To analyze the impact of cecal ligation and puncture (CLP)-induced sepsis on the proliferation and differentiation of intestinal epithelial cells.Methods:① Animal experiment: sixteen male C57BL/6 mice were divided into sham operation group (Sham group) and CLP-induced sepsis model group (CLP group) by random number table method, with 8 mice in each group. After 5 days of operation, the jejunal tissues were taken for determination of leucine-rich-repeat-containing G-protein-coupled receptor 5 (LGR5) and intestinal alkaline phosphatase (IAP) by polymerase chain reaction (PCR). The translation of LGR5 was detected by Western blotting. The expression of proliferating cell nuclear antigen (Ki67) was analyzed by immunohistochemistry. IAP level was detected by modified calcium cobalt staining and colorimetry. Immunofluorescence staining was used to detect the expression of Paneth cell marker molecule lysozyme 1 (LYZ1) and goblet cell marker molecule mucin 2 (MUC2). ② Cell experiment: IEC6 cells in logarithmic growth stage were divided into blank control group and lipopolysaccharide (LPS) group (LPS 5 μg/mL). Twenty-four hours after treatment, PCR and Western blotting were used to analyze the transcription and translation of LGR5. The proliferation of IEC6 cells were detected by 5-ethynyl-2'-deoxyuridine (EdU) staining. The transcription and translation of IAP were detected by PCR and colorimetric method respectively.Results:① Animal experiment: the immunohistochemical results showed that the positive rate of Ki67 staining in the jejunal tissue of CLP group was lower than that of Sham group [(41.7±2.5)% vs. (48.7±1.4)%, P = 0.01]. PCR and Western blotting results showed that there were no statistical differences in the mRNA and protein expressions of LGR5 in the jejunal tissue between the CLP group and Sham group (Lgr5 mRNA: 0.7±0.1 vs. 1.0±0.2, P = 0.11; LGR5/β-actin: 0.83±0.17 vs. 0.68±0.19, P = 0.24). The mRNA (0.4±0.1 vs. 1.0±0.1, P < 0.01) and protein (U/g: 47.3±6.0 vs. 73.1±15.3, P < 0.01) levels of IAP in the jejunal tissue were lower in CLP group. Immunofluorescence saining analysis showed that the expressions of LYZ1 and MUC2 in the CLP group were lower than those in the Sham group. ②Cell experiment: PCR and Western blotting results showed that there was no significant difference in the expression of LGR5 between the LPS group and the blank control group (Lgr5 mRNA: 0.9±0.1 vs. 1.0±0.2, P = 0.33; LGR5/β-actin: 0.71±0.18 vs. 0.69±0.04, P = 0.81). The proliferation rate of IEC6 cells in the LPS group was lower than that in the blank control group, but there was no significant difference [positivity rate of EdU: (40.5±3.8)% vs. (46.5±3.6)%, P = 0.11]. The mRNA (0.5±0.1 vs. 1.0±0.2, P < 0.01) and protein (U/g: 15.0±4.0 vs. 41.2±10.4, P < 0.01) of IAP in the LPS group were lower than those in the blank control group. Conclusion:CLP-induced sepsis inhibits the proliferation and differentiation of intestinal epithelial cells, impairing the self-renewal ability of intestinal epithelium.

Based on the perspective of medical equipment safety culture,analyze the Global Patient Safety Report 2024 released by the World Health Organization(WHO)on May 30,and extract patient safety elements related to medical devices.Propose to initiate action plan for patient safety related to medical devices and discuss the pathway and measures to further ensure patient safety and strengthen safety management in the clinical use of medical devices,in conjunction with promoting high-quality development of hospitals.

OBJECTIVES: To investigate the mechanism of luteolin for inhibiting proliferation of lung cancer A549 cells. METHODS: A549 cells treated with different concentrations of luteolin for 48 h were evaluated for changes in cell viability, proliferation, reactive oxygen species (ROS) production and apoptosis using MTT assay, plate cloning assay, EdU staining, DCFH-DA assay and Hoechst33258 staining. The changes in cell autophagy were examined with MDC staining, and the expressions of apoptosis-related proteins (Bax, Bcl-2, and cleaved caspase-9), autophagy-related proteins (LC3B, Beclin 1, and P62), AKT/mTOR pathway proteins, and HO-1 protein were detected using Western blotting. RESULTS: Treatment with luteolin dose-dependently inhibited the viability and proliferation of A549 cells, increased intracellular ROS levels, up-regulated the expressions of Bax, cleaved caspase-9, and Beclin 1, increased the LC3B-II/LC3B-I ratio, down-regulated the expressions of Bcl-2 and P62, and induced cell apoptosis and autophagy. Luteolin also significantly inhibited the phosphorylation of AKT and mTOR and down-regulated the expression of HO-1 protein in the cells. CONCLUSIONS Luteolin induces apoptosis and autophagy to inhibit proliferation of A549 cells by increasing ROS production, inhibiting the AKT/mTOR pathway and down-regulating HO-1 protein expression.
Humans ; TOR Serine-Threonine Kinases/metabolism* ; A549 Cells ; Reactive Oxygen Species/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Cell Proliferation/drug effects* ; Signal Transduction/drug effects* ; Lung Neoplasms/pathology* ; Apoptosis/drug effects* ; Luteolin/pharmacology* ; Autophagy/drug effects* ; Heme Oxygenase-1/metabolism* ; Cell Survival/drug effects*

Background: Excessive growth of keratinocytes is the critical event in the etiology of psoriasis.However, the underlying molecular mechanism of psoriatic keratinocyte hyperproliferation is still unclear. Objective: This study aimed to figure out the potential contributory role of S-phase kinase-associated protein 2 (SKP2) in promoting the hyperproliferation of keratinocytes in psoriasis. Methods: We analyzed microarray data (GSE41662) to investigate the gene expression of SKP2in psoriatic lesion skins compared with their adjacent non-lesional skin. Then, we further confirmed the mRNA and protein expression of SKP2 in human psoriatic skin tissues, imiquimod (IMQ)-induced psoriatic mice back skins and tumor necrosis factor α (TNF-α), interleukin (IL)-17A and IL-6-stimulated keratinocytes by using real-time quantitative polymerase chain reaction and western blot (WB). Furthermore, we explored the potential pathogenic role and its underlying cellular mechanism of SKP2 in promoting keratinocytes hyperproliferation through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, cell cycle detection, 5-ethynyl-2′-deoxyuridine staining and WB. Finally, we determined whether inhibition of SKP2 can effectively alleviate the keratinocytes hyperproliferation in vivo. Results: We identified that SKP2 is aberrantly upregulated in the psoriatic lesion skin and cytokines-stimulated keratinocytes. Moreover, upregulated SKP2 augments cytokines-induced keratinocytes hyperproliferation. Mechanistically, enhanced SKP2 increased the S phase ratio through inhibiting Cyclin-Dependent Kinase Inhibitor p27 (P27 Kip1) expression. Correspondingly, suppression of SKP2 with SMIP004 can significantly ease the epidermis hyperplasia in vivo. Conclusion Our results suggest that elevated SKP2 can empower keratinocytes proliferation and psoriasis-like epidermis hyperplasia via downregulation of P27 Kip1. Therefore, targeting SKP2-P27 Kip1 axis might be a promising therapeutic strategy for the treatment of psoriasis in future.

Objective:To analyze the clinical features, differential diagnosis, treatment and prognosis of complex lymphatic malformations.Methods:The clinical data of patients with complex lymphatic malformation were retrospectively analyzed from April 2010 to April 2022 in the Multidisciplinary Outpatient Department of the Vascular Disease Team of West China Hospital, Sichuan University. All patients were diagnosed with complex lymphatic malformation after consultation with multidisciplinary experts in pediatric surgery, radiology, plastic surgery, pathology, rehabilitation and other departments. The clinical manifestations, blood routine, coagulation function, magnetic resonance imaging and treatment methods of the patients were analyzed. According to the follow-up and disease results, the patients were divided into improvement, stability, progress and death.Results:A total of 18 patients with complex lymphatic malformations were included in the study, including 6 males and 12 females. The age of first diagnosis ranged from 1 month to 29 years old, and the median age was 2.5 years old. Patients were followed up and treated for 0.4 to 12.0 years, with an average follow-up of 3.5 years. Ten patients had pleural and pericardial effusion; 15 patients had visceral involvement which showed multifocal changes in imaging examinations; 9 cases were accompanied by bone destruction, which in Gorham-Stout disease patients broke through the cortex while in generalized lymphatic anomalies it did not; 14 patients had various degrees of coagulation abnormalities, of which 8 patients with severe coagulation dysfunction were all diagnosed as kaposiform lymphangiomatosis. Of the 18 patients, one kaposiform lymphangiomatosis patient died; six patients progressed; eight patients were stable; and three patients improved.Conclusion:The clinical characteristics of patients with complex lymphatic malformations are systemic, diverse and complex. The clinical symptoms of patients with diffuse lymphatic malformation accompanied by involvement of bone and multiple internal organs, chest and abdominal effusion, and coagulation dysfunction should be considered as complex lymphatic malformation. However, due to overlapping clinical characteristics of each subtypes, it is difficult to distinguish patients with complex lymphatic malformation, and the curative effect and prognosis are poor. Precision targeted drugs are the future research direction for the treatment of such diseases.

Objective:To analyze the clinical features, differential diagnosis, treatment and prognosis of complex lymphatic malformations.Methods:The clinical data of patients with complex lymphatic malformation were retrospectively analyzed from April 2010 to April 2022 in the Multidisciplinary Outpatient Department of the Vascular Disease Team of West China Hospital, Sichuan University. All patients were diagnosed with complex lymphatic malformation after consultation with multidisciplinary experts in pediatric surgery, radiology, plastic surgery, pathology, rehabilitation and other departments. The clinical manifestations, blood routine, coagulation function, magnetic resonance imaging and treatment methods of the patients were analyzed. According to the follow-up and disease results, the patients were divided into improvement, stability, progress and death.Results:A total of 18 patients with complex lymphatic malformations were included in the study, including 6 males and 12 females. The age of first diagnosis ranged from 1 month to 29 years old, and the median age was 2.5 years old. Patients were followed up and treated for 0.4 to 12.0 years, with an average follow-up of 3.5 years. Ten patients had pleural and pericardial effusion; 15 patients had visceral involvement which showed multifocal changes in imaging examinations; 9 cases were accompanied by bone destruction, which in Gorham-Stout disease patients broke through the cortex while in generalized lymphatic anomalies it did not; 14 patients had various degrees of coagulation abnormalities, of which 8 patients with severe coagulation dysfunction were all diagnosed as kaposiform lymphangiomatosis. Of the 18 patients, one kaposiform lymphangiomatosis patient died; six patients progressed; eight patients were stable; and three patients improved.Conclusion:The clinical characteristics of patients with complex lymphatic malformations are systemic, diverse and complex. The clinical symptoms of patients with diffuse lymphatic malformation accompanied by involvement of bone and multiple internal organs, chest and abdominal effusion, and coagulation dysfunction should be considered as complex lymphatic malformation. However, due to overlapping clinical characteristics of each subtypes, it is difficult to distinguish patients with complex lymphatic malformation, and the curative effect and prognosis are poor. Precision targeted drugs are the future research direction for the treatment of such diseases.