RESUMO
Mesenchymal stem cells (MSCs) have pleiotropic immuno-modulatory effects and pro-angiogenic ability, leading to the presumption that MSCs may be involved in the pathogenesis of many inflammatory or autoimmune disorders, including psoriasis. In a previous study, we reported the specific gene expression profile of dermal MSCs from psoriasis. Inflammation- and angiogenesis-related genes, such as lipopolysaccharide-induced tumor necrosis factor-alpha transcription factor (LITAF), dual-specificity protein phosphatase 1 (DUSP1), vascular endothelial growth factor α (VEGFα), and insulin-like growth factor-binding protein-5 (IGFBP5), are abnormally expressed in psoriatic dermal MSCs. As a key regulator of gene expression, miRNA are involved in a wide variety of biological processes; in fact, several miRNAs have been implicated in the development and progression of inflammatory or autoimmune disorders. In this study, we compared the miRNA expression profiles of dermal MSCs from patients with psoriasis to those in MSCs from normal individuals by microarray, and found that the pro-inflammatory miRNA miR-155 was significantly overexpressed in psoriatic MSCs (2.44 fold, P < 0.001). Additionally, the expression of miR-155 target gene TAB2 (8.47 ± 1.55 vs 6.38 ± 2.10, P < 0.01,) and the downstream gene iNOS (5.26 ± 2.58 vs 3.73 ± 1.89, P < 0.05) was found to be inhibited in psoriatic dermal MSCs by real-time PCR. Therefore, we speculated that the elevation in miR-155 levels may be an indicator of, or a key regulatory pathway in, the pathogenesis of psoriasis, resulting in functionally impaired dermal MSCs.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Derme/metabolismo , Regulação da Expressão Gênica , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/genética , Psoríase/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adulto , Estudos de Casos e Controles , Derme/patologia , Feminino , Humanos , Masculino , Células-Tronco Mesenquimais/patologia , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Psoríase/metabolismo , Psoríase/patologia , Reação em Cadeia da Polimerase em Tempo Real , Índice de Gravidade de Doença , Transdução de SinaisRESUMO
Psoriasis is a common chronic relapsing inflammatory skin disease, in which mesenchymal stem cells (MSCs) have been hypothesized to play an important role in abnormal localized inflammation and vascular proliferation observed in skin lesions. Previous studies have revealed abnormal gene expression patterns, DNA methylation status, and cytokine secretion of MSCs in psoriatic skin lesions, as well as some gene expression abnormalities related to inflammation and angiogenesis. We further verified the gene and protein expressions of inflammation-related lipopolysaccharide-induced tumor necrosis factor-alpha transcription factor (LITAF), dual-specificity protein phosphatase 1 (DUSP1), and angiogenesis-related hematopoietically expressed homeobox (HHEX) in MSCs derived from the skin lesions of psoriasis patients. The gene expression of LITAF, DUSP1, and HHEX in dermal MSCs was measured at the mRNA level using reverse transcription-polymerase chain reaction and the corresponding protein expression levels were analyzed by western blotting analysis. The gene and protein expression levels of LITAF, HHEX, and DUSP1 in dermal MSCs were significantly lower in psoriasis patients compared to controls. Amplification and western blotting results were consistent with our previously reported gene chip data. Our results suggest that dermal MSCs in psoriatic skin lesions may be involved in the development, progression, and regulation of localized inflammatory abnormalities by reducing the expression of LITAF, HHEX, and DUSP1, which are related to inflammation and angiogenesis.
Assuntos
Fosfatase 1 de Especificidade Dupla/genética , Expressão Gênica , Proteínas de Homeodomínio/genética , Células-Tronco Mesenquimais/metabolismo , Proteínas Nucleares/genética , Psoríase/genética , Fatores de Transcrição/genética , Adulto , Estudos de Casos e Controles , Fosfatase 1 de Especificidade Dupla/metabolismo , Feminino , Proteínas de Homeodomínio/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Nucleares/metabolismo , Psoríase/diagnóstico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Índice de Gravidade de Doença , Fatores de Transcrição/metabolismo , Adulto JovemRESUMO
Bacillus pumilus is able to secrete subtilisin-like prote-ases, one of which has been purified and characterized biochemically, demonstrating great potential for use in industrial applications. In the current study, the biosynthesis and transcription of extracellular pro-teases in B. pumilus (BA06) under salt stress were investigated using various methods, including a proteolytic assay, zymogram analysis, and real-time PCR. Our results showed that total extracellular proteolytic activity, both in fermentation broth and on milk-containing agar plates, was considerably repressed by salt in a dosage-dependent manner. As Bacillus species usually secret multiple extracellular proteases, a vari-ety of individual extracellular protease encoding genes were selected for real-time PCR analysis. It was shown that proteases encoded by the aprE and aprX genes were the major proteases in the fermentation broth in terms of their transcripts in B. pumilus. Further, transcription of aprE, aprX, and epr genes was indeed repressed by salt stress. In con-trast, transcription of other genes (e.g., vpr and wprA) was not repressed or significantly affected by the salt. Conclusively, salt stress represses total extracellular proteolytic activity in B. pumilus, which can largely be ascribed to suppression of the major protease-encoding genes (aprE, aprX) at the transcriptional level. In contrast, transcription of other pro-tease-encoding genes (e.g., vpr, wprA) was not repressed by salt stress.
Assuntos
Bacillus/enzimologia , Endopeptidases/biossíntese , Estresse Fisiológico/efeitos dos fármacos , Bacillus/efeitos dos fármacos , Endopeptidases/genética , Fermentação , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Cloreto de Sódio/farmacologiaRESUMO
There are significant differences on the biological characteristics of bone marrow mesenchymal stem cells (BMMSCs), immunological response, and antigen-presenting functions between patients with psoriasis and normal subjects, but there are no significant differences in aborted fetuses. We examined the differences in BMMSCs between aborted fetuses and patients with psoriasis in this study. Bone marrow from normal subjects, aborted fetuses, and patients with psoriasis were obtained using a MidiMACS machine. Density gradient centrifugation method was used to isolate the bone marrow mononuclear cells of patients with psoriasis and aborted fetus and the cells were cultivated. Bone marrow CD34(+) cells from normal subjects were isolated. MTT colorimetric detection was used to test the proliferation activity of bone marrow CD34(+) cells. The purity of bone marrow CD34(+) cells and BMMSCs was determined by flow cytometry. The BMMSC culture supernatant fluid of patients with psoriasis and aborted fetuses showed no statistically significant difference with bone marrow CD34(+) cell proliferation in normal subjects (P > 0.05).
Assuntos
Antígenos CD34/metabolismo , Células da Medula Óssea/citologia , Células-Tronco Mesenquimais/citologia , Adulto , Proliferação de Células , Separação Celular , Células Cultivadas , Feminino , Citometria de Fluxo , Humanos , Masculino , Pessoa de Meia-Idade , Psoríase/patologia , Adulto JovemRESUMO
Psoriasis is an inflammatory skin disease characterized by excessive proliferation and abnormal differentiation and apoptosis of keratinocytes (KCs). Mesenchymal stem cells (MSCs) from skin lesions of psoriasis patients demonstrate abnormal cytokine secretion, which may affect KC proliferation and apoptosis. Here, we explored how MSCs from skin lesions of psoriasis patients affect HaCaT cell proliferation and apoptosis. First, flow cytometry and multipotent differentiation methods were used to identify skin MSCs, which were then co-cultured with HaCaT cells. HaCaT cell proliferation was analyzed in real-time, and cell cycle progression and apoptosis were assessed by flow cytometry. Cell morphologies and multipotencies of skin MSCs were similar between the psoriasis group and healthy control group, with high levels of CD29, CD44, CD73, CD90, and CD105 and limited expression of CD34, CD45, and HLA-DR. MSCs from skin lesions of psoriasis patients promote KC proliferation more potently and are less capable of inducing KC apoptosis. This may underlie KC proliferation and abnormal apoptosis in psoriasis skin lesions, which results in abnormal thickening of the epidermis.