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Panax notoginseng saponins (PNS), the active ingredients of the traditional Chinese medicine Panax notoginseng, have strong neuroprotective and anti-platelet aggregation effects. To investigate whether PNS can promote hair follicle growth in C57BL/6J mice, the optimal concentration of PNS was initially determined, followed by clarification of the mechanism underlying their effects. Twenty-five male C57BL/6J mice had the hair on a 2 × 3 cm2 area of the dorsal skin shaved and were equally divided into five groups: control group, 5% minoxidil (MXD) group, and three PNS treatment groups [2% (10 mg/kg), 4% (20 mg/kg), and 8% (40 mg/kg) PNS]. They were then intragastrically administered the corresponding drugs for 28 days. The effects of PNS on C57BL/6J mice were analyzed by subjecting their dorsal depilated skin samples to different assessments, including hematoxylin and eosin staining, immunohistochemistry, immunofluorescence, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blotting (WB). The group with 8% PNS exhibited the largest number of hair follicles from 14 days onwards. Compared with the control group, the number of hair follicles increased significantly in the mice treated with 8% PNS and 5% MXD, which significantly increased in a PNS-dose-dependent manner. Immunohistochemistry and immunofluorescence results revealed that treatment with 8% PNS activated the metabolism of hair follicle cells, with them showing higher rates of proliferation and apoptosis than those in the normal group. In qRT-PCR and WB analysis, the expression of ß-catenin, Wnt10b, and LEF1 was upregulated in the PNS and MDX groups compared with that in the control group. Examination of the WB bands revealed that the greatest inhibitory effect of Wnt5a occurred in mice in the 8% PNS group. PNS may promote the growth of hair follicles in mice, with 8% PNS demonstrating the strongest effect. The mechanism behind this may be related to the Wnt/ß-catenin signaling pathway.
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Panax notoginseng , Saponinas , Camundongos , Masculino , Animais , Folículo Piloso , Saponinas/farmacologia , Via de Sinalização Wnt , Camundongos Endogâmicos C57BLRESUMO
Hair follicle stem cells (HFSCs) are implicated in the formation of hair follicles and epidermis. This study aims to clarify the role of SMAD2 in regulating the differentiation of HFSCs, which is involved with Smurf2. Functional assays were carried out in human HFSCs to assess the effect of SMAD2 and Smurf2 with altered expression on growth dynamics of HFSCs. Ubiquitination of SMAD2 and its protein stability were assessed. The binding relationship between NANOG and DNMT1 was assessed. A mouse skin wound model was induced to verify the effects of Smurf2/SMAD2/NANOG/DNMT1 on wound healing. SMAD2 overexpression was observed in HFSCs during differentiation and its ectopic expression contributed to promotion of differentiation and apoptosis of HFSCs while arresting cell proliferation. Mechanistic investigations indicated that Smurf2 promoted the ubiquitination and degradation of SMAD2, thus causing downregulation of SMAD2 expression. By this mechanism, NANOG expression was reduced and the subsequent DNMT1 transcriptional expression was also diminished, leading to suppression of differentiation and apoptosis of HFSCs while stimulating cell proliferation. Moreover, in vivo data showed that Smurf2 upregulation limited epidermal wound healing in mice by inhibiting the SMAD2/NANOG/DNMT1 axis. Our work proposed a potential target regarding SMAD2 restoration in promoting HFSC differentiation and skin wound healing.
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INTRODUCTION: Adult stem cell function has been one of the most intensively explored areas of biological and biomedical research, with hair follicle stem cells serving as one of the best model systems. This study explored the role of the transcription factor DLX5 in regulating hair follicle stem cell (HFSC) differentiation. METHODS: HFSCs were isolated, characterized, and assessed for their expression of DLX5, c-MYC, NSD1, and miR-29c-3p using RT-qPCR, Western blot analysis, or immunofluorescence. Next, the ability of HFSCs to proliferate as well as differentiate into either sebaceous gland cells or epidermal cells was determined. The binding of DLX5 to the c-MYC promoter region, the binding of c-MYC to the miR-29c-3p promoter region, and the binding of miR-29c-3p to the 3'-UTR of NSD1 mRNA were verified by luciferase activity assay and ChIP experiments. RESULTS: DLX5 was highly expressed in differentiated HFSCs. DLX5 transcriptionally activated c-MYC expression to induce HFSC differentiation. c-MYC was able to bind the miR-29c-3p promoter and thus suppressed its expression. Without miR-29c-3p mediated suppression, NSD1 was then able to promote HFSC differentiation. These in vitro experiments suggested that DLX5 could promote HFSC differentiation via the regulation of the c-MYC/miR-29c-3p/NSD1 axis. DISCUSSION: This study demonstrates that DLX5 promotes HFSC differentiation by modulating the c-MYC/miR-29c-3p/NSD1 axis and identifies a new mechanism regulating HFSC differentiation.
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OBJECTIVE: To observe the clinical efficacy of thunder-fire moxibustion combined with mifepristone for ovarian chocolate cyst dysmenorrhea with kidney deficiency and blood stasis. METHODS: Seventy patients were randomly divided into an observation group and a control group, 35 cases in each group. The patients in the the control group were treated with oral administration of mifepristone, 10 mg each time, once a day; based on the treatment of the control group, the patients in the observation group were treated with thunder-fire moxibustion at Guanyuan (CV 4), Zigong (EX-CA 1), Xuehai (SP 10), once every other day. Both the groups were treated for 3 months. The Cox menstrual symptom scale (CMSS) score, the maximum cross-sectional area of ectopic cyst, and the serum levels of transforming growth factor-ß1 (TGF-ß1) and interleukin-17 (IL-17) were observed before and after treatment in the two groups. The clinical efficacy was evaluated. RESULTS: Compared before treatment, the severity scores and duration scores of CMSS as well as the serum levels of TGF-ß1 were reduced after treatment in the two groups (P<0.05), and the serum level of IL-17 in the observation group was reduced (P<0.05); the reducing of the severity score and duration score of CMSS as well as the serum levels of TGF-ß1 and IL-17 in the observation group were more significant than those in the control group (P<0.05). After treatment, the maximum cross-sectional area of ectopic cyst in the two groups was decreased (P<0.05), and the reducing in the observation group was more significant than that in the control group (P<0.05). The total effective rate was 94.3% (33/35) in the observation group, which was higher than 71.4% (25/35) in the control group (P<0.05). CONCLUSION: Thunder-fire moxibustion combined with mifepristone could significantly improve dysmenorrhea symptoms, shorten dysmenorrhea time and promote atrophy of ovarian heterotopic cyst in patients with ovarian chocolate cyst dysmenorrhea of kidney deficiency and blood stasis, and the mechanism may be related to the reduction of serum levels of TGF-ß1 and IL-17.
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Chocolate , Cistos , Moxibustão , Pontos de Acupuntura , Dismenorreia/tratamento farmacológico , Feminino , Humanos , Rim , MifepristonaRESUMO
Hypoxia is of great significance for stem cells to maintain the proliferation and differentiation capacity. As a specialized mesenchymal component of the hair follicle (HF), the dermal papilla cell (DPC) not only regulates HF cycle, but also plays a pivotal role in differentiating hair follicle stem cell(HFSC) into HF. However, whether hypoxia could affect DPCs on proliferation or metabolism remains unclear. In our study, DPCs were cultured in normoxia (20%O2 ) or hypoxia (5%O2 ). Cell viability assays were performed, and lactate dehydrogenase (LDH) activity and lactate level in DPCs were detected. After that, LDH was overexpressed or knocked down in DPCs; then, the expression of protein markers (ALP, Ki-67) was assessed by Western blotting, and cell proliferation was also detected after overexpression or knockdown of LDH. Hypoxia did show positive effect on proliferation of DPCs. The LDH activity of DPCs cultured under hypoxic condition was significantly higher than that of cultured under normoxic condition. Overexpression of LDH significantly up-regulates the expression of ALP and Ki-67 compared with knockdown and negative control. Cell proliferation was also promoted in DPCs with elevated LDH. Our findings showed that the proliferation activity of DPCs could be stimulated under hypoxia. Meanwhile, LDH plays an important role in maintaining the activity of DPCs in hypoxic condition.
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Folículo Piloso , L-Lactato Desidrogenase , Proliferação de Células , Células Cultivadas , Humanos , HipóxiaRESUMO
It is well known that dermal papilla cells (DPCs) are crucial for hair follicle growth and regeneration. However, dermal papilla cells in 2D culture could lose their ability of regeneration after several passage intervals. As opposed to DPCs in 2D culture, the DPCs in 3D culture could passage extensively. However, the molecular mechanisms of DPCs' regeneration in 3D culture remain unclear. Accordingly, gene sequencing is recommended for the investigation of hair regeneration between 2D and 3D culture, the three groups were established including DPCs in passage 2 in 2D culture, DPCs in passage 8 in 2D culture and DPCs in passage 8 in 3D culture. The differentially expressed genes (DEGs) were identified using the Venn diagram of these three groups, which included 1642 known and 359 novel genes, respectively. A total of 1642 known genes were used for Gene Ontology (GO), Kyoto Gene, Genomic Encyclopedia (KEGG) pathway enrichment and protein-protein interaction (PPI) analyses, respectively. The functions and pathways of DEGs were enriched in biological regulation, signal transduction and immune system, etc. The key module and the top 10 hub genes (IL1B, CXCL12, HGF, EGFR, APP, CCL2, PTGS2, MMP9, NGF and SPP1) were also identified using the Cytoscape application. Furthermore, the qRT-PCR results of the three groups validated that the hub genes were crucial for hair growth. In conclusion, the ten identified hub genes and related pathways in the current study can be used to understand the molecular mechanism of hair growth, and those provided a possibility for hair regeneration.
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Derme/citologia , Folículo Piloso/citologia , Folículo Piloso/fisiologia , Regeneração , Análise de Sequência de RNA , Técnicas de Cultura de Células , Proliferação de Células , Células Cultivadas , Biologia Computacional/métodos , Derme/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Ontologia Genética , Redes Reguladoras de Genes , Humanos , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas , Esferoides CelularesRESUMO
BACKGROUND: Alopecia is a highly prevalent disease characterizing by the loss of hair. Dermal papilla (DP) cells are the inducer of hair follicle regeneration, and in vitro three-dimensional (3D) culturing DP cells have been proven to induce hair follicle regeneration. However, the molecular mechanisms behind the regulation of 3D culturing DP cells remain unclear. METHODS: 3D-cultivated DP cells were used as in vitro cell model. DP sphere xenograft to nude mice was performed for in vivo study of hair follicle regeneration. qRT-PCR, Western blotting, and immunofluorescence were used for detecting the level of XIST, miR-424 and Hedgehog pathway-related proteins, respectively. H&E staining was used to examine hair neogenesis. Cell viability, proliferation and ALP activity were measured by MTT, CCK-8 and ELISA assays, respectively. Luciferase assays were used for studying molecular regulation between XIST, miR-424 and Shh 3'UTR. RESULTS: XIST and Shh were up-regulated, and miR-424 was down-regulated in 3D DP cells. Molecular regulation studies suggested that XIST sponged miR-424 to promote Shh expression. Knockdown of XIST suppressed DP cell activity, cell proliferation, ALP activity and the expression of other DP markers by sponging miR-424. Knockdown of XIST suppressed Shh mediated hedgehog signaling by targeting miR-424. Moreover, the knockdown of XIST inhibited DP sphere induced in vivo hair follicle regeneration and hair development. CONCLUSION: XIST sponges miR-424 to promote Shh expression, thereby activating hedgehog signaling and facilitating DP mediated hair follicle regeneration.
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Derme/metabolismo , Folículo Piloso/fisiologia , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , Regeneração/fisiologia , Transdução de Sinais , Fosfatase Alcalina/metabolismo , Animais , Sequência de Bases , Proliferação de Células/genética , Sobrevivência Celular/genética , Células Cultivadas , Proteínas Hedgehog/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Camundongos Nus , RNA Longo não Codificante/genética , Esferoides Celulares/metabolismoRESUMO
BACKGROUND: The failure of hair follicle regeneration is the major cause of alopecia, which is a highly prevalent disease worldwide. Dermal papilla (DP) cells play important role in the regulation of hair follicle regeneration. However, the molecular mechanism of how dermal papilla cells direct follicle regeneration is still to be elucidated. METHODS: In vitro DP 3D culturing and in vivo nude mice DP sphere implanted models were used to examine the molecular regulation of DP cells and follicle regeneration. qRT-PCR and Western blotting were used to detect gene and protein expression, respectively. Immunofluorescence was used to detect the expression level of Wnt10b, Ki-67 and ß-catenin. Luciferase assay was used to examine the relationship among PCAT1, miR-329 and Wnt10b. ALP activity was measured by ELISA. H&E staining was used to measure follicle growth in skin tissues. RESULTS: Up-regulation of PCAT1 and Wnt10b, however, down-regulation of miR-329 were found in the in vitro 3D dermal papilla. Bioinformatics analysis and luciferase assays demonstrated that PCAT1 promoted Wnt10b expression by sponging miR-329. Knockdown of PCAT1 suppressed the proliferation and activity, as well as ALP and other DP markers of DP cells by targeting miR-329. Knockdown of PCAT1 regulated miR-329/Wnt10b axis to attenuate ß-catenin expression and nucleus translocation to inhibit Wnt/ß-catenin signaling. Furthermore, knockdown of PCAT1 suppressed DP sphere induced follicle regeneration and hair growth in nude mice. CONCLUSION: PCAT1 maintains characteristics of DP cells by targeting miR-329 to activating Wnt/ß-catenin signaling pathway, thereby promoting hair follicle regeneration.
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Folículo Piloso/crescimento & desenvolvimento , MicroRNAs/genética , RNA Longo não Codificante/genética , Regeneração/genética , Animais , Proliferação de Células/genética , Proliferação de Células/fisiologia , Regulação para Baixo , Genes Neoplásicos/genética , Humanos , Camundongos Nus , Proteínas Proto-Oncogênicas/metabolismo , RNA Longo não Codificante/metabolismo , Regeneração/fisiologia , Pele/metabolismo , Regulação para Cima , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/genéticaRESUMO
OBJECTIVE: To investigate the effect of natural hirudin on revascularization of ischemic skin flap in rats using Micro-CT and three-dimensional (3D) reconstruction. METHODS: Thirty-two Sprague Dawley rats were prepared a ischemic skin flap (8.0 cm×1.8 cm) model on the back and randomly divided into hirudin group and control group (16 rats in each group). At immediate and within 3 days after operation, the rats were treated with hypodermic injection of natural hirudin 0.3 mL (including natural hirudin 6 ATU) every day in hirudin group and the equal amount of normal saline in control group. At 6 days after operation, the survival rate of skin flap was evaluated, histological changes were observed by HE staining, and the volemia, length of blood vessels, and number of blood vessels were analyzed with Micro-CT 3D reconstruction. RESULTS: Both groups of rats survived to the end of the experiment without infection. Different degrees of necrosis occurred in the distal part of the skin flaps in both groups at 6 days after operation, but the flap survival rate of the hirudin group (72.11%±8.97%) was significantly higher than that of control group (58.94%±4.02%) ( t=3.280, P=0.008). Histological observation showed that the histological hierarchy of the hirudin group was clearer than that of the control group, with more microangiogenesis and less inflammatory response and inflammatory cell infiltration. Micro-CT 3D reconstruction showed that the flap vessels in the hirudin group were more and denser, and the volemia, length of blood vessels, and number of blood vessels were significantly higher than those in the control group ( P<0.05). CONCLUSION: Natural hirudin can reduce the inflammation of tissue, promote the regeneration and recanalization of blood vessels in ischemic skin flap, so as to improve the survival rate of the flap.
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Sobrevivência de Enxerto , Terapia com Hirudina , Transplante de Pele , Pele/irrigação sanguínea , Animais , Inflamação , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Microtomografia por Raio-XRESUMO
Malignant melanoma is one of the most common types of cancer worldwide. Efforts have been made to elucidate the pathology of malignant melanoma. However, its molecular mechanisms remain unclear. Therefore, the microarray datasets GSE3189, GSE4570 and GSE4587 from the Gene Expression Omnibus database were used for the elucidation of candidate genes involved in the initiation and progression of melanoma. Assessment of the microarray datasets led to the identification of differentially expressed genes (DEGs), which were subsequently used for function enrichment analysis. These data were utilized in the construction of the protein-protein interaction network and module analysis was conducted using STRING and Cytoscape software. The results of these analyses led to the identification of a total of 182 DEGs, including 52 downregulated and 130 upregulated genes. The functions and pathways found to be enriched in the DEGs were GTPase activity, transcription from RNA polymerase II promoter, apoptotic processes, cell adhesion, membrane related pathways, calcium signaling cascade and the PI3K-Akt signaling pathway. The identified genes were demonstrated to belong to a set of 10 hub genes biologically involved in proliferation, apoptosis, cytokinesis, adhesion and migration. Survival analysis and Oncomine database analysis revealed that the calmodulin gene family, BAX and VEGFA genes, may be associated with the initiation, invasion or recurrence of melanoma. In conclusion, the DEGs and hub genes identified in the present study may be used to understand the molecular pathways involved in the initiation and progression of malignant melanoma. Furthermore, the present study may aid in the identification of possible targets for the diagnosis and treatment of melanoma.
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PURPOSE: To investigate whether hirudin exerts its antithrombin action to decrease the ratio of Human Microvascular Endothelial Cells (HMVECs) apoptosis. METHODS: Human microvascular endothelial cells (HMVECs) cultured in the third and fifth generations were used. HMVECs were divided into normal group, thrombin group (T group), natrual hirudin group (H group), thrombin + natrual hirudin group (T + H group), AG490 group, thrombin + AG490 group (T + AG490 group), natrual hirudin + AG490 group (H + AG490 group), thrombin + natural hirudin + AG490 (T + H + AG490 group).Apart from the normal group, the other groups were exposed to the relevant drugs for 24 hours.HMVEC apoptosis was assessed by flow cytometric and double Immunofluorescence of phosphorylation of JAK (P-JAK2) and TUNEL assay. RESULTS: Compared with the normal group, in thrombin group the HMVECs apoptosis rate were significantly increased (P<0.05).The results indicated that the index of apoptosis and the apoptosis rate were improved in cultures treated by natural hirudin (T + H group), relative to cultures with thrombin only (T group). We found that the index of apoptosis and the apoptosis rate in the AG490 + thrombin group were higher than that in the hirudin + thrombin group (P<0.05). Double Immunofluorescence of p-JAK2 and TUNEL assays showed that cells were double positive for P-JAK2 uptake and TUNEL detection liquid binding. CONCLUSION: The natural hirudin and JAK2/STATs signal inhibitor AG490 could block the effects of thrombin. Natural hirudin could attenuate HMVECs apoptosis via antagonizing thrombin and it is suggested that this effect may occur by blocking the JAK2/STATs signaling pathway and this signaling pathways appears to be not the only pathway.
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Antitrombinas/farmacologia , Apoptose/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Hirudinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Trombina/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Humanos , Microvasos/efeitos dos fármacos , Microvasos/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Abstract Purpose: To investigate whether hirudin exerts its antithrombin action to decrease the ratio of Human Microvascular Endothelial Cells (HMVECs) apoptosis. Methods: Human microvascular endothelial cells (HMVECs) cultured in the third and fifth generations were used. HMVECs were divided into normal group, thrombin group (T group), natrual hirudin group (H group), thrombin + natrual hirudin group (T + H group), AG490 group, thrombin + AG490 group (T + AG490 group), natrual hirudin + AG490 group (H + AG490 group), thrombin + natural hirudin + AG490 (T + H + AG490 group).Apart from the normal group, the other groups were exposed to the relevant drugs for 24 hours.HMVEC apoptosis was assessed by flow cytometric and double Immunofluorescence of phosphorylation of JAK (P-JAK2) and TUNEL assay. Results: Compared with the normal group, in thrombin group the HMVECs apoptosis rate were significantly increased (P<0.05).The results indicated that the index of apoptosis and the apoptosis rate were improved in cultures treated by natural hirudin (T + H group), relative to cultures with thrombin only (T group). We found that the index of apoptosis and the apoptosis rate in the AG490 + thrombin group were higher than that in the hirudin + thrombin group (P<0.05). Double Immunofluorescence of p-JAK2 and TUNEL assays showed that cells were double positive for P-JAK2 uptake and TUNEL detection liquid binding. Conclusion: The natural hirudin and JAK2/STATs signal inhibitor AG490 could block the effects of thrombin. Natural hirudin could attenuate HMVECs apoptosis via antagonizing thrombin and it is suggested that this effect may occur by blocking the JAK2/STATs signaling pathway and this signaling pathways appears to be not the only pathway.
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Humanos , Trombina/efeitos dos fármacos , Antitrombinas/farmacologia , Hirudinas/farmacologia , Apoptose/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Microvasos/efeitos dos fármacos , Microvasos/metabolismoRESUMO
Objective: To explore the effect of natural hirudin on proliferation of human microvascular endothelial cells (HMVECs) and its preliminary mechanism of promoting angiogenesis. Methods: Three-dimensional culture models of HMVECs were established in vitro and observed by inverted phase contrast microscopy after 24 hours of culturing. Then, the three-dimensional culture models of HMVECs were treated with different concentrations (1, 4, and 7 ATU/mL) of the natural hirudin, respectively, and Dulbecco's modified Eagle's medium containing 10% fetal bovine serum as control. The cell proliferations of 4 groups were detected by cell counting kit 8 (CCK-8) method at 24, 48, and 72 hours; the angiogenesis of 4 groups were observed by tube formation assay at 24 hours; the expressions of vascular endothelial growth factor (VEGF) and Notch1 of HMVECs in 4 groups were observed by immunofluorescence staining at 24 hours. Results: The observation of cells in three-dimensional culture models showed that HMVECs attached to Matrigel well, and the cells formed tube structure completely after 24 hours. The results of CCK-8 test showed that the absorbance ( A) value of 1 and 4 ATU/mL groups were higher than that of control group at each time point ( P<0.05), and A value of 4 ATU/mL group was the highest. The A value of 7 ATU/mL group was significantly lower than those of 1 and 4 ATU/mL groups and control group ( P<0.05). The tube formation assay showed that the tube structure was more in 1 and 4 ATU/mL groups than in 7 ATU/mL group and control group, and in 4 ATU/mL group than in 1 ATU/mL group, showing significant differences ( P<0.05). There was no significant difference between 7 ATU/mL group and control group ( P>0.05). The results of immunofluorescence staining showed that compared with control group, the Notch1 expression was higher in 1 and 4 ATU/mL groups and lower in 7 ATU/mL group; and there was significant difference between 4 and 7 ATU/mL groups and control group ( P<0.05). The VEGF expression was higher in 1, 4, and 7 ATU/mL groups than in control group, in 4 ATU/mL group than in 1 and 7 ATU/mL groups, showing significant differences ( P<0.05). Conclusion: Natural hirudin can promote angiogenesis at low and medium concentrations, but suppress angiogenesis at high concentrations. Its mechanism may be related to the VEGF-Notch signal pathway.
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Células Endoteliais , Hirudinas , Neovascularização Fisiológica , Fator A de Crescimento do Endotélio Vascular , Proliferação de Células , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Hirudinas/fisiologia , Humanos , Neovascularização Fisiológica/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Macrostomia (Tessier's 7 cleft) is a rare congenital lip deformity. Macrostomia can occur unilateral or bilateral, isolated or associated with other syndromes. Isolated bilateral macrostomia is exceedingly rare with only a few cases reported to date. The authors report 6 cases of isolated bilateral macrostomia surgically repaired in 4-layered approaches. The traditional method was improved and the result obtained was satisfactory after longest follow-up of 3 years. The technique is easy to imitate, simple in design, aesthetically and functionally corrects the deformity.
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Macrostomia/cirurgia , Procedimentos Cirúrgicos Bucais/métodos , Procedimentos de Cirurgia Plástica/métodos , Retalhos Cirúrgicos , Pré-Escolar , Feminino , Humanos , Lactente , Macrostomia/diagnóstico , Masculino , Mucosa Bucal/transplanteRESUMO
Objective: To investigate the effect of natural hirudin combined with hyperbaric oxygen therapy on the survival of transplanted random-pattern skin flap in rats. Methods: A random-pattern skin flap in size of 10.0 cm×2.5 cm was elevated on the dorsum of 72 Sprague Dawley rats. Then the 72 rats were randomly divided into 4 groups ( n=18) according to the therapy method. At immediate and within 4 days after operation, the rats were treated with normal saline injection in control group, normal saline injection combined with hyperbaric oxygen treatment in hyperbaric oxygen group, the natural hirudin injection in natural hirudin group, and the natural hirudin injection combined with hyperbaric oxygen treatment in combined group. The flap survival was observed after operation, and survival rate was evaluated at 6 days after operation. The skin samples were collected for histological analysis, microvessel density (MVD) measurement, and evaluation of tumor necrosis factor α (TNF-α) expression level by the immunohistochemical staining at 2 and 4 days after operation. Results: Partial necrosis occurred in each group after operation, and the flap in combined group had the best survival. The survival rate of flap was significantly higher in hyperbaric oxygen group, natural hirudin group, and combined group than that in control group, and in combined group than in hyperbaric oxygen group and natural hirudin group ( P<0.05). There was no significant difference between hyperbaric oxygen group and natural hirudin group ( P>0.05). At 2 days, more microvascular structure was observed in hyperbaric oxygen group, natural hirudin group, and combined group in comparison with control group; while plenty of inflammatory cells infiltration in all groups. At 4 days, the hyperbaric oxygen group, natural hirudin group, and the combined group still showed more angiogenesis. Meanwhile, there was still infiltration of inflammatory cells in control group, inflammatory cells in the other groups were significantly reduced when compared with at 2 days. At 2 days, the MVD was significantly higher in hyperbaric oxygen group, natural hirudin group, and combined group than that in control group ( P<0.05); the expression of TNF-α was significantly lower in hyperbaric oxygen group, natural hirudin group, and combined group than that in control group ( P<0.05). There was no significant difference in above indexes between hyperbaric oxygen group, natural hirudin group, and combined group ( P>0.05). At 4 days, the MVD was significantly higher in hyperbaric oxygen group, natural hirudin group, and combined group than that in control group, in natural hirudin group and combined group than in hyperbaric oxygen group ( P<0.05). The expression of TNF-α was significantly lower in hyperbaric oxygen group, natural hirudin group, and combined group than that in control group, in combined group than in natural hirudin group and hyperbaric oxygen group ( P<0.05). Conclusion: Hyperbaric oxygen and natural hirudin therapy after random-pattern skin flap transplantation can improve the survival of flaps. Moreover, combined therapy is seen to exhibit significant synergistic effect. This effect maybe related to promotion of angiogenesis and the reduction of inflammation response.
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Antitrombinas/farmacocinética , Sobrevivência de Enxerto/efeitos dos fármacos , Hirudinas/farmacologia , Oxigenoterapia Hiperbárica , Transplante de Pele , Pele/efeitos dos fármacos , Retalhos Cirúrgicos , Animais , Inflamação , Necrose , Ratos , Ratos Sprague-Dawley , Pele/metabolismo , Retalhos Cirúrgicos/irrigação sanguínea , Retalhos Cirúrgicos/fisiologia , Fator de Transcrição RelA/metabolismo , Transplantes , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Cell surface engineering technology advances cell therapeutics and tissue engineering by accurate micro/nanoscale control in cell-biomaterial ensembles and cell spheroids formation. By tailoring cell surface, microgels can encapsulate cells for versatile uses. However, microgels are coated in a thick layer to house multiple cells together but not a single cell based. Besides, excessive deposition on cell surface is detrimental to cellular functions. Herein, layer-by-layer (LbL) self-assembly to encapsulate single cell using nanogel is reported, owing to its security and tunable thickness at nanoscale, and further forms cell spheroids by physical cross-linking on nanogel-coated cells for delivery. A hair follicle (HF) regeneration model where the dermal papilla cells (DPCs) are given a 3D installation to maintain its ability of HF induction during in vitro culture is studied. Dermal papilla (DP) spheroids are optimized and that LbL-DPCs aggregation is akin to primary DP is demonstrated. The markers ALP, Versican, and NCAM are examined to investigate that high-passaged (P8) DP spheroids can restore the hair induction potential, which are lost in 2D culture. New HFs are regenerated successfully by implantation of DP spheroids in vivo.
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Folículo Piloso/citologia , Esferoides Celulares/citologia , Engenharia Tecidual/métodos , Animais , Folículo Piloso/metabolismo , Humanos , Esferoides Celulares/metabolismoRESUMO
BACKGROUND: The efficiency of hair follicle (HF) reconstruction is decreased by extensive apoptotic remodeling that occurs soon after grafting. OBJECTIVE: To evaluate a basement membrane matrix (matrix) to improve the efficiency of HF reconstruction and serve as a cell delivery vehicle. MATERIALS AND METHODS: Newborn mouse skin cells were suspended in a matrix and transplanted in a chamber assay. The viability and proliferation of mouse dermal papilla cells seeded in the matrix were tested. Dermal papilla cells and epidermal cells seeded in matrix sheets were grafted into nude mice to observe hair formation. RESULTS: The matrix significantly shortened the time to hair formation. The first hair shafts appeared within the matrix at 17.67 ± 1.21 days versus 23.00 ± 1.41 days for Dulbecco's modified Eagle medium controls. There was a significant difference (p < .05) in the number of newly formed hairs in areas of reconstructed skin with the matrix (100 µL) grafts (323 ± 12) versus controls (276 ± 11). Dermal papilla cells were successfully cultured in the matrix, and hair formation was dense when the matrix was used as a cell delivery vehicle for follicle reconstruction. CONCLUSION: The matrix improved the efficiency of HF reconstruction and was a suitable delivery vehicle of cells for HF engineering.
Assuntos
Folículo Piloso/fisiologia , Folículo Piloso/cirurgia , Regeneração , Animais , Membrana Basal/citologia , Membrana Basal/fisiologia , Transplante de Células , Células Cultivadas , Derme/citologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
According to the growth state of hair follicles, the hair cycle is divided into the anagen, catagen and telogen phases. A number of biological factors have been shown to synchronize with the hair cycle. As an important component of the hair follicle, the extracellular matrix is regulated by matrix metalloproteinases (MMPs) and their inhibitors (tissue inhibitor of matrix metalloproteinases; TIMPs). It has been reported that MMP-2, MMP-9 and TIMP-1 are associated with the hair cycle; however, their expression levels during the hair cycle have not been fully elucidated. Reverse transcription-polymerase chain reaction and ELISA analysis in the present study demonstrated that, during the hair cycle in mice, mRNA and protein expression levels of MMP-2 and MMP-9 were elevated in the anagen phase, and decreased during the catagen and telogen phases. Furthermore, SDS-PAGE gelatin zymography demonstrated that their activities fluctuated in the hair cycle. Additionally, it was observed that the mRNA and protein expression levels of TIMP-1 and TIMP-2 were negatively correlated with MMP-9 and MMP-2, respectively. Immunohistochemical examination demonstrated that MMP-2 and TIMP-2 were present in all structures of the hair follicle. However, MMP-9 and TIMP-1 were locally expressed in certain areas of the hair follicle, such as in the sebaceous gland at the anagen, catagen and telogen phases, and in the inner root sheath at the catagen phase. These results suggested that MMP-2 and MMP-9 may serve an important role in the hair growth cycle.
RESUMO
A strategy utilizing elastin peptide macroporous cryogels to build highly flexible scaffolds to load carbon nanotubes, polypyrrole, and iron oxide magnetic nanoparticles, is presented. This combines high elasticity, flexibility, shape memory property, and injectable property together with conductivity and/or magnetic responsive property. The network can afford 97.5% compressive strain with an excellent conductivity of 50.1 ± 2.9 S cm(-1) at 90% strain.
RESUMO
Human dermal papilla (DP) cells have been studied extensively when grown in the conventional monolayer. However, because of great deviation from the real in vivo three-dimensional (3D) environment, these two-dimensional (2D) grown cells tend to lose the hair-inducible capability during passaging. Hence, these 2D caused concerns have motivated the development of novel 3D culture techniques to produce cellular microtissues with suitable mimics. The hanging-drop approach is based on surface tension-based technique and the interaction between surface tension and gravity field that makes a convergence of liquid drops. This study used this technique in a converged drop to form cellular spheroids of dermal papilla cells. It leads to a controllable 3Dspheroid model for scalable fabrication of inductive DP microtissues. The optimal conditions for culturing high-passaged (P8) DP spheroids were determined first. Then, the morphological, histological and functional studies were performed. In addition, expressions of hair-inductive markers including alkaline phosphatase, α-smooth muscle actin and neural cell adhesion molecule were also analyzed by quantitative RT-PCR, immunostaining and immunoblotting. Finally, P8-DP microtissues were coimplanted with newborn mouse epidermal cells (EPCs) into nude mice. Our results indicated that the formation of 3D microtissues not only endowed P8-DP microtissues many similarities to primary DP, but also confer these microtissues an enhanced ability to induce hair-follicle (HF) neogenesis in vivo. This model provides a potential to elucidate the native biology of human DP, and also shows the promising for the controllable and scalable production of inductive DP cells applied in future follicle regeneration.