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1.
Methods Mol Biol ; 2269: 175-201, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33687680

RESUMO

Bench-to-bedside axis of therapeutic product development is currently being oriented towards minimum invasiveness on both ends-not only clinical application but harvesting of the starting biological material as well. This is particularly relevant for Advanced Therapy Medicinal Products and their specific legislative requirements, even more so in skin regeneration. It is precisely the skin equivalents and grafts that benefit from the minimum-to-noninvasive approach to a noteworthy extent, taking in account the sensitive nature of both skin harvesting and grafting.This chapter includes protocols for two separate steps of generating skin equivalent from the cells cultured from hair follicle outer root sheath. The first step is a non-pigmented epidermal equivalent generated from human keratinocytes from the outer root sheath named non-pigmented epidermal graft. The second step consists of co-cultivating human keratinocytes and human melanocytes from the outer root sheath, hereby producing a pigmented epidermal graft.


Assuntos
Derme/metabolismo , Fibroblastos/metabolismo , Folículo Piloso/metabolismo , Queratinócitos/metabolismo , Melanócitos/metabolismo , Engenharia Tecidual , Técnicas de Cocultura , Derme/citologia , Fibroblastos/citologia , Folículo Piloso/citologia , Humanos , Queratinócitos/citologia , Melanócitos/citologia
2.
J Nat Med ; 75(2): 326-338, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33417145

RESUMO

A methanol extract from Isodonis Herba demonstrated significant proliferative effect on human hair follicle dermal papilla cells (HFDPC, % of control: 150.0 ± 2.0% at 20 µg/mL, p < 0.01). From the extract, 14 ent-kaurane-type diterpenoids (1-14), two abietane-type diterpenoids (15 and 16) and four triterpenoids (17-20) were isolated. Among the isolates, enmein (1, 160.9 ± 3.0% at 20 µM, p < 0.01), isodocarpin (2, 169.3 ± 4.9% at 5 µM, p < 0.01), nodosin (4, 160.5 ± 12.4% at 20 µM, p < 0.01), and oridonin (8, 165.4 ± 10.6% at 10 µM, p < 0.01) showed the proliferative effects. The principal component enmein (1) activated the expression of vascular endothelial growth factor (VEGF) mRNA, upregulated the production of VEGF and increased levels of phospho-Akt, phospho-GSK-3ß, and ß-catenin accumulation in HFDPC, which could be the mechanism of these activate proliferation activity.


Assuntos
Diterpenos de Caurano/metabolismo , Folículo Piloso/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , beta Catenina/metabolismo , Proliferação de Células , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos
3.
Gene ; 770: 145339, 2021 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-33333220

RESUMO

Hair follicle (HF) development is characterized by periodic growth cycles regulated by numerous factors. We previously showed that SMAD2 might be involved in the HF growth cycle in Angora rabbits. However, its extra role in the HF growth and development remains obscure. In this study, we cloned the complete coding sequence (CDS) of the Angora rabbit SMAD2 gene. Within SMAD2 CDS, we identified the open reading frame (ORF) had a length of 1314 bp and encoding 437 amino acids. Bioinformatics analyses revealed that the SMAD2 protein is unstable and hydrophilic, and predominatelylocalizesin the cell nucleus. We identified that SMAD2 expression was elevated in the telogen phase of the during HF cycle. The knockdown and overexpression of SMAD2 could regulate HF growth and development related genes, such as WNT2, FGF2, and LEF1.Furthermore, SMAD2 may upregulate TGF-ß signaling pathway-related genes, including TFDP1, E2F4, and RBL1. In conclusion, our results indicate that SMAD2 plays a vital role in HF development by regulating the TGF-ß signaling pathway.


Assuntos
Folículo Piloso/metabolismo , Proteína Smad2/metabolismo , Animais , Fator 2 de Crescimento de Fibroblastos/metabolismo , Folículo Piloso/citologia , Masculino , Coelhos , Proteína p107 Retinoblastoma-Like/metabolismo , Proteína Wnt2/metabolismo
4.
Gene ; 771: 145343, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33333231

RESUMO

Annexin A1 (ANXA1), a calcium-dependent phospholipid binding protein expressed in animals, plants and microorganisms, participates in various cellular physiological activities. Previous proteomics analysis indicates that the level of ANXA1 in mice dorsal skin changes during hair growth cycle, we speculate that ANXA1 may play an important role in hair follicle (HF) development. Thus, Anxa1 knock-out (KO) and over-expression (OE) mice were constructed to test its function. Our results showed that in addition to the diameter of HF and hair shaft, ANXA1 could participate in hair growth by affecting the density of HF, and the proliferation of hair follicle stem cells (HFSCs). Meanwhile, molecular analysis showed that EGF signaling pathway is involved in the function of ANXA1. The expression of Anxa1 is negatively correlated with the levels of Egf, Notch1, Mkk7, and phosphorylated AKT1 and ERK/2 proteins. The levels of Egf, Notch1, Mkk7 and phosphorylation of AKT1 and ERK/2 increased in Anxa1 KO mice but decreased in Anxa1 OE mice. Taken together, our results suggested that ANXA1 could affect the hair growth by regulating the HFSCs proliferation through EGF signaling pathway.


Assuntos
Anexina A1/genética , Anexina A1/metabolismo , Folículo Piloso/crescimento & desenvolvimento , Animais , Proliferação de Células , Fator de Crescimento Epidérmico/metabolismo , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Folículo Piloso/metabolismo , Camundongos , Fosforilação , Transdução de Sinais
5.
Methods Mol Biol ; 2202: 51-61, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32857345

RESUMO

Reactive oxygen species (ROS) may severely affect the biochemical viability of most cells. However, ROS may act also as key second messengers regulating important physiological functions in eukaryotic organisms. Of special interest is the potential role of ROS in the regulation of stem cell function and tissue homeostasis and regeneration in adult mammalian tissues. In this context, the hair follicle constitutes an excellent experimental model to study this aspect of ROS biology.Here we present a robust protocol to promote a sustained growth of ex vivo cultured human hair follicles based on the induction of a transient/modulable production of nonlethal endogenous ROS levels in the tissue through a protoporphyrin IX-dependent photodynamic procedure. The light-switchable ROS production activates hair follicle stem cell niches, induces cell proliferation, and maintains the growth/anagen phase for long time. This approach constitutes a complementary experimental tool to study the physiological roles of ROS in human tissues.


Assuntos
Técnicas de Cultura de Células/métodos , Folículo Piloso/crescimento & desenvolvimento , Espécies Reativas de Oxigênio/metabolismo , Adulto , Proliferação de Células/fisiologia , Células Cultivadas , Cabelo/fisiologia , Folículo Piloso/metabolismo , Humanos , Nicho de Células-Tronco/fisiologia , Células-Tronco/fisiologia
6.
PLoS One ; 15(12): e0243507, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33351808

RESUMO

OBJECTIVE: Mature hair follicles represent an important stage of hair follicle development, which determines the stability of hair follicle structure and its ability to enter the hair cycle. Here, we used weighted gene co-expression network analysis (WGCNA) to identify hub genes of mature skin and hair follicles in Inner Mongolian cashmere goats. METHODS: We used transcriptome sequencing data for the skin of Inner Mongolian cashmere goats from fetal days 45-135 days, and divided the co expressed genes into different modules by WGCNA. Characteristic values were used to screen out modules that were highly expressed in mature skin follicles. Module hub genes were then selected based on the correlation coefficients between the gene and module eigenvalue, gene connectivity, and Gene Ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. The results were confirmed by quantitative polymerase chain reaction (qPCR). RESULTS: Ten modules were successfully defined, of which one, with a total of 3166 genes, was selected as a specific module through sample and gene expression pattern analyses. A total of 584 candidate hub genes in the module were screened by the correlation coefficients between the genes and module eigenvalue and gene connectivity. Finally, GO/KEGG functional enrichment analyses detected WNT10A as a key gene in the development and maturation of skin hair follicles in fetal Inner Mongolian cashmere goats. qPCR showed that the expression trends of 13 genes from seven fetal skin samples were consistent with the sequencing results, indicating that the sequencing results were reliable.n.


Assuntos
Cabras/genética , Folículo Piloso/embriologia , Animais , China , Desenvolvimento Fetal/genética , Feto/metabolismo , Perfilação da Expressão Gênica/métodos , Ontologia Genética , Redes Reguladoras de Genes/genética , Genoma/genética , Cabras/embriologia , Folículo Piloso/metabolismo , RNA Mensageiro/genética , Pele/metabolismo , Transcriptoma/genética
7.
Nat Commun ; 11(1): 5079, 2020 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-33033234

RESUMO

Tumor heterogeneity and lack of knowledge about resistant cell states remain a barrier to targeted cancer therapies. Basal cell carcinomas (BCCs) depend on Hedgehog (Hh)/Gli signaling, but can develop mechanisms of Smoothened (SMO) inhibitor resistance. We previously identified a nuclear myocardin-related transcription factor (nMRTF) resistance pathway that amplifies noncanonical Gli1 activity, but characteristics and drivers of the nMRTF cell state remain unknown. Here, we use single cell RNA-sequencing of patient tumors to identify three prognostic surface markers (LYPD3, TACSTD2, and LY6D) which correlate with nMRTF and resistance to SMO inhibitors. The nMRTF cell state resembles transit-amplifying cells of the hair follicle matrix, with AP-1 and TGFß cooperativity driving nMRTF activation. JNK/AP-1 signaling commissions chromatin accessibility and Smad3 DNA binding leading to a transcriptional program of RhoGEFs that facilitate nMRTF activity. Importantly, small molecule AP-1 inhibitors selectively target LYPD3+/TACSTD2+/LY6D+ nMRTF human BCCs ex vivo, opening an avenue for improving combinatorial therapies.


Assuntos
Carcinoma Basocelular/metabolismo , Proteínas Hedgehog/metabolismo , Transdução de Sinais , Neoplasias Cutâneas/metabolismo , Fator de Transcrição AP-1/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Cromatina/metabolismo , DNA de Neoplasias/metabolismo , Resistencia a Medicamentos Antineoplásicos , Matriz Extracelular/metabolismo , Ontologia Genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Folículo Piloso/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Proteínas de Neoplasias/metabolismo , Ligação Proteica , Proteína Smad3/metabolismo , Transativadores/metabolismo , Regulação para Cima
8.
Nat Cell Biol ; 22(6): 640-650, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32393888

RESUMO

Tissue homeostasis and regeneration rely on resident stem cells (SCs), whose behaviour is regulated through niche-dependent crosstalk. The mechanisms underlying SC identity are still unfolding. Here, using spatiotemporal gene ablation in murine hair follicles, we uncover a critical role for the transcription factors (TFs) nuclear factor IB (NFIB) and IX (NFIX) in maintaining SC identity. Without NFI TFs, SCs lose their hair-regenerating capability, and produce skin bearing striking resemblance to irreversible human alopecia, which also displays reduced NFIs. Through single-cell transcriptomics, ATAC-Seq and ChIP-Seq profiling, we expose a key role for NFIB and NFIX in governing super-enhancer maintenance of the key hair follicle SC-specific TF genes. When NFIB and NFIX are genetically removed, the stemness epigenetic landscape is lost. Super-enhancers driving SC identity are decommissioned, while unwanted lineages are de-repressed ectopically. Together, our findings expose NFIB and NFIX as crucial rheostats of tissue homeostasis, functioning to safeguard the SC epigenome from a breach in lineage confinement that otherwise triggers irreversible tissue degeneration.


Assuntos
Alopecia/patologia , Diferenciação Celular , Cromatina/metabolismo , Folículo Piloso/citologia , Fatores de Transcrição NFI/fisiologia , Células-Tronco/citologia , Alopecia/genética , Alopecia/metabolismo , Animais , Células Cultivadas , Cromatina/genética , Feminino , Folículo Piloso/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regeneração , Células-Tronco/metabolismo
9.
PLoS One ; 15(4): e0231376, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32298297

RESUMO

Hair follicle stem cells (HFSCs) have been shown to be essential in the development and regeneration of hair follicles (HFs). The Inner Mongolia Cashmere goat (Capra hircus) has two types of HFs, primary and secondary, with cashmere being produced from the secondary hair follicle. To identify the genes associated with cashmere growth, transcriptome profiling of anagen and telogen secondary HFSCs was performed by RNA-Seq. The RNA-Seq analysis generated over 58 million clean reads from each group, with 2717 differentially expressed genes (DEGs) detected between anagen and telogen, including 1500 upregulated and 1217 downregulated DEGs. A large number of DEGs were predominantly associated with cell part, cellular process, binding, biological regulation and organelle. In addition, the PI3K-Akt, MAPK, Ras and Rap1 signaling pathways may be involved in the growth of HFSCs cultured in vitro. The RNA-Seq results showed that the well-defined HFSC signature genes and cell cycle-associated genes showed no significant differences between anagen and telogen HFSCs, indicating a relatively quiescent cellular state of the HFSCs cultured in vitro. These results are useful for future studies of complex molecular mechanisms of hair follicle cycling in cashmere goats.


Assuntos
Cabras/genética , Folículo Piloso/citologia , Células-Tronco Mesenquimais/metabolismo , Transcriptoma , Animais , Células Cultivadas , Perfilação da Expressão Gênica/métodos , Perfilação da Expressão Gênica/normas , Folículo Piloso/metabolismo , RNA-Seq/métodos , Lã/citologia , Lã/metabolismo
10.
PLoS One ; 15(4): e0232206, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32330194

RESUMO

Hair growth is the cyclically regulated process that is characterized by growing phase (anagen), regression phase (catagen) and resting phase (telogen). Hair follicle stem cells (HFSCs) play pivotal role in the control of hair growth cycle. It has been notified that stem cells have the distinguished metabolic signature compared to differentiated cells, such as the preference to glycolysis rather than mitochondrial respiration. Crif1 is a mitochondrial protein that regulates the synthesis and insertion of oxidative phosphorylation (OXPHOS) polypeptides to inner membrane of mitochondria. Several studies demonstrate that tissue-specific knockout of Crif1 leads to mitochondrial dysfunction. In this study, we investigated the effect of mitochondrial dysfunction in terms of Crif1 deficiency on the hair growth cycle of adult mice. We created two kinds of inducible conditional knockout (icKO) mice. In epidermal specific icKO mice (Crif1 K14icKO), hair growth cycle was significantly retarded compared to wild type mice. Similarly, HFSC specific icKO mice (Crif1 K15icKO) showed significant retardation of hair growth cycle in depilation-induced anagen model. Interestingly, flow cytometry revealed that HFSC populations were maintained in Crif1 K15icKO mice. These results suggest that mitochondrial function in HFSCs is important for the progression of hair growth cycle, but not for maintenance of HFSCs.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Folículo Piloso/crescimento & desenvolvimento , Folículo Piloso/metabolismo , Cabelo/crescimento & desenvolvimento , Cabelo/metabolismo , Animais , Diferenciação Celular/fisiologia , Epiderme/crescimento & desenvolvimento , Epiderme/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Fosforilação Oxidativa , Peptídeos/metabolismo , Células-Tronco/metabolismo
11.
Sci Rep ; 10(1): 4519, 2020 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-32161290

RESUMO

Inner Mongolia cashmere goats, as an important part of animal husbandry production, play an important role in animal fiber industry. In recent years, scientific research has made a lot of explorations on the molecular regulation mechanism of hair follicle cycle growth, but few studies have been reported on the development of cashmere hair in fetal period. This study was based on the completion of 21 skin samples of mRNA and miRNA sequencing in 7 fetal periods (45 days, 55 days,65 days,75 days,95 days,115 days and 135 days) of the Inner Mongolia Cashmere goat. The target genes of miRNA associated with the development of secondary hair follicles in the cashmere goats were selected through the combination analysis of mRNA and miRNA data. Then the overexpression vector was constructed and the interaction between the miRNA and the target gene was identified by Dual-Luciferase Reporter Gene System. The function and interaction relationship of chi-miR-199a-5p and TGF-ß2 were verified by RT-qPCR and western blot at the level of the fibroblasts in Inner Mongolia Cashmere goat. It provides a theoretical basis for further study of miRNA and its target genes regulating the occurrence and development of skin hair follicles. As the result shows, the expression trends of 7 genes (BAMBI, SMAD1, LTBP1, PPP2R1B, ID4, BMP8B and PITX2) and 7 miRNA (chi-miR-17-5p, chi-miR-125b-3p, chi-miR-21-5p, chi-miR-143-5p and chi-miR-106b-5p) in the skin samples for the seven stages of the fetus were shown to be consistent with the sequencing results. the results of sequencing are reliable. The correlation coefficient of TGF-ß2 and chi-miR-199a-5p in fetal 45d-135d expression is -0.84, showing a strong negative correlation, The target relationship was preliminarily judged. The results of double luciferase vector report showed that chi-miR-199a-5p significantly decreased the expression of luciferase in TGF-ß2 3'UTR, It is determined that there is a reciprocal relationship between them at a specific time. We transfected chi-miR199a-5p-FAM mimics into fibroblasts cultured in vitro from Inner Mongolia cashmere goats. After transfection, the cells were harvested to extract total RNA and protein. The mRNA and protein expression levels of TGF-ß2 in fibroblasts were detected by RT-qPCR and western blot. It was verified that chi-miR-199a-5p inhibited TGF-ß2 expression at both mRNA and protein translation levels in fibroblasts. At the same time, it was again proved that the TGF-ß2 gene is a target gene of chi-miR199a-5p.


Assuntos
Regulação da Expressão Gênica , Cabras/genética , Folículo Piloso/metabolismo , MicroRNAs/genética , Interferência de RNA , RNA Mensageiro/genética , Animais , Biologia Computacional/métodos , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Ontologia Genética , Genes Reporter , Sequenciamento de Nucleotídeos em Larga Escala
12.
Sci Rep ; 10(1): 4887, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32184439

RESUMO

Cellular metabolism is one of the crucial factors to regulate epigenetic landscape in various cells including immune cells, embryonic stem cells and hair follicle stem cells. Dermal papilla cells (DP) interact with epithelial stem cells to orchestrate hair formation. Here we show that active DP exhibit robust aerobic glycolysis. We observed decrease of signature genes associated with hair induction by DP in presence of low glucose (2 mM) and glycolysis inhibitors. Moreover, hair shaft elongation was attenuated by glycolysis inhibitors. Interestingly, excessive glucose is able to increase the expression of hair inductive genes and elongation of hair shaft. We also observed glycolysis-mediated histone acetylation is increased and chemical inhibition of acetyltransferase reduces expression of the signature genes associated with hair induction in active DP. These results suggest that glucose metabolism is required for expression of signature genes associated with hair induction. This finding may be beneficial for establishing and maintaining of active DP to generate hair follicle in vitro.


Assuntos
Derme/metabolismo , Glucose/metabolismo , Folículo Piloso/metabolismo , Histonas/metabolismo , Acetilação , Animais , Western Blotting , Sobrevivência Celular/fisiologia , Carboidratos da Dieta/metabolismo , Feminino , Glicólise/fisiologia , Metanálise como Assunto , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo Real
13.
PLoS One ; 15(3): e0230380, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32163511

RESUMO

Epidermal morphogenesis and hair follicle (HF) development depend on the ability of keratinocytes to adhere to the basement membrane (BM) and migrate along the extracellular matrix. Integrins are cell-matrix receptors that control keratinocyte adhesion and migration, and are recognized as major regulators of epidermal homeostasis. How integrins regulate the behavior of keratinocytes during epidermal morphogenesis remains insufficiently understood. Here, we show that α-parvin (α-pv), a focal adhesion protein that couples integrins to actin cytoskeleton, is indispensable for epidermal morphogenesis and HF development. Inactivation of the murine α-pv gene in basal keratinocytes results in keratinocyte-BM detachment, epidermal thickening, ectopic keratinocyte proliferation and altered actin cytoskeleton polarization. In vitro, α-pv-null keratinocytes display reduced adhesion to BM matrix components, aberrant spreading and stress fibers formation, and impaired directed migration. Together, our data demonstrate that α-pv controls epidermal homeostasis by facilitating integrin-mediated adhesion and actin cytoskeleton organization in keratinocytes.


Assuntos
Membrana Basal/metabolismo , Epiderme/crescimento & desenvolvimento , Folículo Piloso/metabolismo , Queratinócitos/metabolismo , Proteínas dos Microfilamentos/fisiologia , Morfogênese/fisiologia , Actinas/metabolismo , Animais , Membrana Basal/citologia , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Células Cultivadas , Adesões Focais/metabolismo , Integrinas/metabolismo , Queratinócitos/citologia , Camundongos , Camundongos Transgênicos
14.
Int J Mol Sci ; 21(7)2020 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-32218218

RESUMO

Increasing cashmere yield is one of the important goals of cashmere goat breeding. To achieve this goal, we screened the key genes that can improve cashmere performance. In this study, we used the RNA raw datasets of the skin and dermal papilla cells of secondary hair follicle (SHF-DPCs) samples of hair follicle (HF) anagen and telogen of Albas cashmere goats and identified a set of significant differentially expressed genes (DEGs). To explore potential associations between gene sets and SHF growth features and to identify candidate genes, we detected functional enrichment and constructed protein-protein interaction (PPI) networks. Through comprehensive analysis, we selected Thymosin ß4 (Tß4), Rho GTPase activating protein 6 (ARHGAP6), ADAM metallopeptidase with thrombospondin type 1 motif 15, (ADAMTS15), Chordin (CHRD), and SPARC (Osteonectin), cwcv and kazal-like domains proteoglycan 1 (SPOCK1) as candidate genes. Gene set enrichment analysis (GSEA) for these genes revealed Tß4 and ARHGAP6 have a close association with the growth and development of SHF-DPCs. However, the expression of Tß4 in the anagen was higher than that in the telogen, so we finally chose Tß4 as the ultimate research object. Overexpressing Tß4 promoted and silencing Tß4 inhibited the proliferation of SHF-DPCs. These findings suggest that Tß4 can promote the growth and development of SHF-DPCs and indicate that this molecule may be a valuable target for increasing cashmere production.


Assuntos
Proliferação de Células , Folículo Piloso/metabolismo , Timosina/metabolismo , Animais , Células Cultivadas , Ativação Enzimática , Perfilação da Expressão Gênica , Cabras , Folículo Piloso/citologia , Folículo Piloso/crescimento & desenvolvimento , Timosina/genética
15.
Sci Rep ; 10(1): 1693, 2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-32015359

RESUMO

This study was designed to characterize the location, morphology and ultrastructure of telocytes (TCs) in human scalp tissue. After obtaining approval for this study and informed consent from the patient, a scalp specimen was obtained. The distribution and morphology of TCs in human scalp tissue was assessed by immunohistochemical staining of CD34 and CD117/c-KIT, and the ultrastructure of TCs was investigated using transmission electron microscopy (TEM). Immunohistochemical staining of CD34 revealed that TCs were located in the connective tissue of human scalp, and were concentrated around hair follicles (HFs), blood vessels, sweat glands, sebaceous glands and adipose lobules. Immunohistochemical staining of CD117 revealed that TCs were mainly located in the dermis of human scalp, surrounding the HFs and sweat glands. Under TEM, TCs were seen and confirmed by their special morphological features. These cells were spindle-shaped, had small cell bodies and long thin processes, and surrounded stem cell clusters in the bulge region of HFs. These results demonstrate that TCs in human scalp were positive for CD34 and CD117, and their strategic positioning surrounding stem cells suggests their possible involvement in local regeneration, remodeling and homeostasis of the skin.


Assuntos
Tecido Adiposo/fisiologia , Folículo Piloso/metabolismo , Couro Cabeludo/metabolismo , Glândulas Sudoríparas/fisiologia , Telócitos/fisiologia , Adulto , Antígenos CD34/metabolismo , Folículo Piloso/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imuno-Histoquímica/métodos , Masculino , Microscopia Eletrônica de Transmissão , Proteínas Proto-Oncogênicas c-kit/metabolismo , Regeneração , Couro Cabeludo/ultraestrutura , Adulto Jovem
16.
Acta Histochem ; 122(2): 151484, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31902536

RESUMO

The aim of this study was to evaluate whether the addition of synthetic polymers to the vitrification solution affected follicular morphology and development and the expression of Ki-67, Aquaporin 3 (AQP3) and cleaved Caspase-3 proteins in ovarian tissue of the caprine species. Caprine ovaries were fragmented and two fragments were immediately fixed (Fresh Control) for morphological evaluation, while other two were in vitro cultured for 7 days (Cultured Control) and fixed as well. The remaining fragments were distributed in two different vitrification groups: Vitrified and Vitrified/Cultured. Each group was composed of 4 different treatments: 1) Sucrose (SUC); 2) SuperCool X-1000 0.2 % (X-1000); 3) SuperCool Z-1000 0.4 % (Z-1000) or 4) with polyvinylpyrrolidone K-12 0.2 % (PVP). Also, Fresh Control, Cultured Control, SUC and X-1000 were destined to immunohistochemical detection of Ki-67, AQP3 and cleaved Caspase-3 proteins. Morphologically, the treatment with X-1000 showed no significant difference with the Fresh Control group and was superior to the other treatments. After the cleaved caspase-3 analysis, X-1000 showed the lowest percentages of strong immunostaining while Cultured Control showed the highest. Also, a positive correlation was found between the percentages of degenerated follicles and the percentages of strong staining intensity follicles. Regarding the AQP3 analysis, the highest percentages of strong AQP3 staining intensity were found in X-1000. In conclusion, we have demonstrated that the addition of the synthetic polymer SuperCool X-1000 to the vitrification solution improved the current vitrification protocol of caprine ovarian tissue.


Assuntos
Oócitos/efeitos dos fármacos , Folículo Ovariano/efeitos dos fármacos , Ovário/efeitos dos fármacos , Polímeros/farmacologia , Animais , Criopreservação/métodos , Feminino , Cabras , Folículo Piloso/efeitos dos fármacos , Folículo Piloso/metabolismo , Oócitos/metabolismo , Ovário/metabolismo , Técnicas de Cultura de Tecidos/métodos , Vitrificação
17.
Sci Rep ; 10(1): 176, 2020 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-31932640

RESUMO

Clobetasol propionate (CLO) is a potent glucocorticoid used to treat inflammation-based skin, scalp, and hair disorders. In such conditions, hair follicles (HF) are not only the target site but can also act as drug reservoirs when certain formulations are topically applied. Recently, we have demonstrated nanostructured lipid carriers (NLC) containing CLO presenting epidermal-targeting potential. Here, the focus was evaluating the HF uptake provided by such nanoparticles in comparison to a commercial cream and investigating the influence of different physical stimuli [i.e., infrared (IR) irradiation (with and without metallic nanoparticles-MNP), ultrasound (US) (with and without vibration) and mechanical massage] on their follicular targeting potential. Nanosystems presented sizes around 180 nm (PdI < 0.2) and negative zeta potential. The formulation did not alter skin water loss measurements and was stable for at least 30 days at 5 °C. Nanoparticles released the drug in a sustained fashion for more than 3 days and increased passively about 40 times CLO follicular uptake compared to the commercial cream. Confocal images confirmed the enhanced follicular delivery. On the one hand, NLC application followed by IR for heat generation showed no benefit in terms of HF targeting even at higher temperatures generated by metallic nanoparticle heating. On the other hand, upon US treatment, CLO retention was significantly increased in deeper skin layers. The addition of mechanical vibration to the US treatment led to higher follicular accumulation compared to passive exposure to NLC without stimuli. However, from all evaluated stimuli, manual massage presented the highest follicular targeting potential, driving more than double the amount of CLO into the HF than NLC passive application. In conclusion, NLC showed great potential for delivering CLO to HF, and a simple massage was capable of doubling follicular retention.


Assuntos
Clobetasol/administração & dosagem , Portadores de Fármacos/química , Folículo Piloso/metabolismo , Lipídeos/química , Nanopartículas/administração & dosagem , Absorção Cutânea , Pele/metabolismo , Clobetasol/química , Folículo Piloso/efeitos dos fármacos , Humanos , Raios Infravermelhos , Nanopartículas/química , Pele/efeitos dos fármacos , Estresse Mecânico , Ultrassom
19.
Int J Mol Sci ; 21(3)2020 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-31991762

RESUMO

Despite advances in medical treatments, the proportion of the population suffering from alopecia is increasing, thereby creating a need for new treatments to control hair loss and prevent balding. Human hair follicle dermal papilla cells (hDPCs), a type of specialized fibroblast in the hair bulb, play an essential role in controlling hair growth and in conditions like androgenic alopecia. This study aimed to evaluate the intensity-dependent effect of extremely low-frequency electromagnetic fields (ELF-EMFs) on the expression of anagen-related molecules in hDPCs in vitro. We examined the effect of ELF-EMF on hDPCs to determine whether activation of the GSK-3ß/ERK/Akt signaling pathway improved hDPC activation and proliferation; hDPCs were exposed to ELF-EMFs at a frequency of 70 Hz and at intensities ranging from 5 to 100 G, over four days. Various PEMF intensities significantly increased the expression of anagen-related molecules, including collagen IV, laminin, ALP, and versican. In particular, an intensity of 10 G is most potent for promoting the proliferation of hDPC and expression of anagen-related molecules. Moreover, 10 G ELF-EMF significantly increased ß-catenin and Wnt3α expression and GSK-3ß/ERK/Akt phosphorylation. Our results confirmed that ELF-EMFs enhance hDPC activation and proliferation via the GSK-3ß/ERK/Akt signaling pathway, suggesting a potential treatment strategy for alopecia.


Assuntos
Campos Eletromagnéticos , Regulação da Expressão Gênica/efeitos da radiação , Glicogênio Sintase Quinase 3 beta/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos da radiação , Biomarcadores , Proliferação de Células , Células Cultivadas , Derme/citologia , MAP Quinases Reguladas por Sinal Extracelular , Folículo Piloso/citologia , Folículo Piloso/metabolismo , Humanos , Fosforilação , Via de Sinalização Wnt/efeitos da radiação
20.
Sci Rep ; 10(1): 454, 2020 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-31949201

RESUMO

The periodic regrowth of rabbit fur is economically important. Here, we aimed to characterise the histological traits and microRNA (miRNA) expression profiles in the skin tissue of Wan Strain Angora rabbits at different weeks after plucking. Haematoxylin-eosin staining showed that hair follicles were in the telogen phase in the first week, while they were in the anagen phase from the fourth to twenty-fourth weeks. In addition, two small RNA libraries derived from skin samples of Wan Strain Angora rabbits at telogen and anagen stages yielded over 24 million high-quality reads. Specifically, 185 miRNAs were differentially expressed between the telogen and anagen phases. The function of the differentially expressed miRNAs was explored by comparing them with known mammalian miRNAs and by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis of their predicted targets. Five new functional miRNAs were validated using quantitative real-time PCR. Moreover, the fibroblast growth factor 5 (FGF5) gene was verified to be a target of conservative_NC_013672.1_9290 and conservative_NC_013675.1_10734. We investigated differential miRNA profiles between the telogen and anagen phases of the hair cycle and our findings provide a basis for future studies focusing on the mechanisms of miRNA-mediated regulation of rabbit hair follicle cycling.


Assuntos
MicroRNAs/genética , Pele/crescimento & desenvolvimento , Pele/metabolismo , Animais , Folículo Piloso/metabolismo , Anotação de Sequência Molecular , Coelhos
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