Gene cloning and sequence analysis of the RPL29 gene and its effect on lipogenesis in goat intramuscular adipocytes / 生物工程学报

The aim of this study was to clone the goat RPL29 gene and analyze its effect on lipogenesis in intramuscular adipocytes. Using Jianzhou big-eared goats as the object, the goat RPL29 gene was cloned by reverse transcription-polymerase chain reaction (RT-PCR), the gene structure and expressed protein sequence were analyzed by bioinformatics, and the mRNA expression levels of RPL29 in various tissues and different differentiation stages of intramuscular adipocytes of goats were detected by quantitative real-time PCR (qRT-PCR). The RPL29 overexpression vector pEGFP-N1-RPL29 constructed by gene recombination was used to transfect into goat intramuscular preadipocytes and induce differentiation. Subsequently, the effect of overexpression of RPL29 on fat droplet accumulation was revealed morphologically by oil red O and Bodipy staining, and changes in the expression levels of genes related to lipid metabolism were detected by qRT-PCR. The results showed that the length of the goat RPL29 was 507 bp, including a coding sequence (CDS) region of 471 bp which encodes 156 amino acid residues. It is a positively charged and stable hydrophilic protein mainly distributed in the nucleus of cells. Tissue expression profiling showed that the expression level of this gene was much higher in subcutaneous adipose tissue and inter-abdominal adipose tissue of goats than in other tissues (P < 0.05). The temporal expression profile showed that the gene was expressed at the highest level at 84 h of differentiation in goat intramuscular adipocytes, which was highly significantly higher than that in the undifferentiated period (P < 0.01). Overexpression of RPL29 promoted lipid accumulation in intramuscular adipocytes, and the optical density values of oil red O staining were significantly increased (P < 0.05). In addition, overexpression of RPL29 was followed by a highly significant increase in ATGL and ACC gene expression (P < 0.01) and a significant increase in FASN gene expression (P < 0.05). In conclusion, the goat RPL29 may promote intra-muscular adipocyte deposition in goats by up-regulating FASN, ACC and ATGL.

Overexpression of chicken Klf2 promotes klf7 transcription and inhibits adipocyte differentiation / 生物工程学报

Overexpression of Krüppel like factor 2 (Klf2) or Klf7 inhibits adipocyte formation. However, it remains unclear whether Klf2 regulates klf7 expression in adipose tissue. In this study, oil red O staining and Western blotting were employed to study the effect of Klf2 overexpression on the differentiation of chicken preadipocytes. The results showed that Klf2 overexpression inhibited the differentiation of chicken preadipocytes induced by oleate and the expression of pparγ, while promoted klf7 expression in chicken preadipocytes. Spearman correlation analysis was used to study the correlation between the expression data of klf2 and klf7 in the adipose tissue of both human and chicken. The results showed that there was a significantly positive correlation between the expression of klf2 and klf7 in adipose tissues (r > 0.1). Luciferase reporter assay showed that overexpression of Klf2 significantly promoted the activity of chicken klf7 promoter (-241/-91, -521/-91, -1 845/-91, -2 286/-91, -1 215/-91; P < 0.05). In addition, the activity of klf7 promoter (-241/-91) reporter in chicken preadipocytes was significantly positively correlated with the amount of klf2 overexpression plasmid transfected (Tau=0.917 66, P=1.074×10-7). Moreover, Klf2 overexpression significantly promoted the mRNA expression of klf7 in chicken preadipocytes (P < 0.05). In conclusion, upregulation of klf7 expression might be one of the pathways that Klf2 inhibits chicken adipocyte differentiation, and the sequence from -241 bp to -91 bp upstream chicken klf7 translation start site might mediate the regulation of Klf2 on klf7 transcription.

Research advances of adipose stem cell matrix gel in promoting wound healing / 中华烧伤杂志

In recent years, with the problem of aging population in China being prominant, the number of patients with chronic wounds such as diabetic foot, pressure ulcer, and vascular ulcer is increasing. Those diseases seriously affect the life quality of patients and increase the economy and care burden of the patients' family, which have been one of the most urgent clinical problems. Many researches have confirmed that adipose stem cells can effectively promote wound healing, while exogenous protease is needed, and there are ethical and many other problems, which limit the clinical application of adipose stem cells. Adipose stem cell matrix gel is a gel-like mixture of biologically active extracellular matrix and stromal vascular fragment obtained from adipose tissue by the principle of fluid whirlpool and flocculation precipitation. It contains rich adipose stem cells, hematopoietic stem cells, endothelial progenitor cells, and macrophages, etc. The preparation method of adipose stem cell matrix gel is simple and the preparation time is short, which is convenient for clinical application. Many studies at home and abroad showed that adipose stem cell matrix gel can effectively promote wound healing by regulating inflammatory reaction, promoting microvascular reconstruction and collagen synthesis. Therefore, this paper summarized the preparation of adipose stem cell matrix gel, the mechanism and problems of the matrix gel in promoting wound repair, in order to provide new methods and ideas for the treatment of chronic refractory wounds in clinic.

Bone Marrow Adipocytes Promote the Survival of Multiple Myeloma Cells and Up-Regulate Their Chemoresistance / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of adipocytes in the bone marrow microenvironment of patients with multiple myeloma (MM) on the pathogenesis of MM.@*METHODS@#Bone marrow adipocytes (BMA) in bone marrow smears of health donors (HD) and newly diagnosed MM (ND-MM) patients were evaluated with oil red O staining. The mesenchymal stem cells (MSC) from HD and ND-MM patients were isolated, and in vitro co-culture assay was used to explore the effects of MM cells on the adipogenic differentiation of MSC and the role of BMA in the survival and drug resistance of MM cells. The expression of adipogenic/osteogenic differentiation-related genes PPAR-γ, DLK1, DGAT1, FABP4, FASN and ALP both in MSC and MSC-derived adipocytes was determined with real-time quantitative PCR. The Western blot was employed to detect the expression levels of IL-6, IL-10, SDF-1α, TNF-α and IGF-1 in the supernatant with or without PPAR-γ inhibitor.@*RESULTS@#The results of oil red O staining of bone marrow smears showed that BMA increased significantly in patients of ND-MM compared with the normal control group, and the BMA content was related to the disease status. The content of BMA decreased in the patients with effective chemotherapy. MM cells up-regulated the expression of MSC adipogenic differentiation-related genes PPAR-γ, DLK1, DGAT1, FABP4 and FASN, but the expression of osteogenic differentiation-related gene ALP was significantly down-regulated. This means that the direct consequence of the interaction between MM cells and MSC in the bone marrow microenvironment is to promote the differentiation of MSC into adipocytes at the expense of osteoblasts, and the cytokines detected in supernatant changed. PPAR-γ inhibitor G3335 could partially reverse the release of cytokines by BMA. Those results confirmed that BMA regulated the release of cytokines via PPAR-γ signal, and PPAR-γ inhibitor G3335 could distort PPAR-γ mediated BMA maturation and cytokines release. The increased BMA and related cytokines effectively promoted the proliferation, migration and drug resistance of MM cells.@*CONCLUSION@#The BMA and its associated cytokines are the promoting factors in the survival, proliferation and migration of MM cells. BMA can protect MM cells from drug-induced apoptosis and plays an important role in MM treatment failure and disease progression.

Hipertrofia y resistencia a la insulina en un modelo in vitro de obesidad y DMT2 inducida por alta glucosa e insulina / Hypertrophy and insulin resistance in an in vitro model of obesity and T2DM induced by high glucose and insulin

Resumen Introducción: La obesidad es considerada un factor de riesgo para desarrollar resistencia a la insulina. La expansión del tejido adiposo se ha relacionado con el aumento de la producción de citoquinas proinflamatorias que, junto a los ácidos grasos son responsables, al menos en parte, del desarrollo de la resistencia a la insulina y esta a su vez, facilita el desarrollo de diabetes mellitus tipo 2 (DMT2). Objetivo: El propósito de este estudio fue realizar y caracterizar un modelo in vitro de obesidad empleando concentraciones altas de glucosa e insulina en una línea de células adipocitarias. Métodos: Se indujo modelo de hipertrofia celular realizando un estímulo en adipocitos maduros con una concentración de glucosa (450 mg/dL) e insulina (106 pmol/L) (modelo HGHI). Tras estímulo se realizaron ensayos de viabilidad celular, diámetro celular, movilización de lípidos y marcadores de señalización de insulina. Resultados: Tras el tratamiento con HGHI, se evidencia hipertrofia adipocitaria, incremento en la acumulación de lípidos, reducción de la ruptura de éstos, alteración de la señalización de insulina y tendencia a modificación de proteínas de marcadores de estrés de retículo y estrés oxidativo. Conclusión: Estos resultados demuestran la validez del modelo in vitro que simula al menos en parte la obesidad asociada a insulino resistencia, siendo una herramienta útil para estudiar los mecanismos de susceptibilidad a obesidad y resistencia a la insulina inducida in vitro con diferentes moléculas.

Abstract Introduction: Obesity is considered a risk factor for developing insulin resistance. The increase in adipose tissue has been related to the increase in the production of pro-inflammatory cytokines, which together with fatty acids are responsible, at least in part, for the development of insulin resistance, and this in turn facilitates the development of T2 diabetes mellitus type 2 (DMT2). Objective: The purpose of this study was to perform and characterize an in vitro model of obesity using high concentrations of glucose and insulin on an adipocyte cell line. Methods: A cell hypertrophy model was induced by stimulating mature adipocytes with a concentration of glucose (450 mg/dL) and insulin (106 pmol/L) (HGHI model). The cell viability, cell diameter, lipid mobilization and insulin signalling markers were evaluated. Results: After HGHI treatment, adipocytes show hypertrophy, increase in lipid accumulation, reduction of lipid breakdown, alteration of insulin signalling, a tendency to modify proteins of reticulum stress markers and, oxidative stress. Conclusion: These results demonstrate a new in vitro model that simulates, at least in part, obesity associated with insulin resistance being a useful tool to study the mechanisms of susceptibility to obesity and insulin resistance induced in vitro by different molecules.

Identification of new human mesenchymal stem cell biomarker by aptamers / Identificação de novos biomarcadores de células tronco mesenquimais de origem humana por meio de aptâmeros

Stem cells are undifferentiated cells that can be distinguished from others by their ability to self-renew and to differentiate into new specific cell types. Mesenchymal stem cells (MSC) are adult stem cells that can be obtained from different sources, such as adipose tissue, bone marrow, dental pulp, and umbilical cord. They can either replicate, originating new identical cells, or differentiate into cells of mesodermal origin and from other germ layers. MSC have been studied as new tools for regenerative therapy. Although encouraging results have been demonstrated, MSC-based therapies still face a great barrier: the difficulty of isolating these cells from heterogeneous environments. MSC are currently characterized by immunolabelling through a set of multiple surface membrane markers, including CD29, CD73, CD90 and CD105, which are also expressed by other cell types. Hence, the present work aimed to identify new specific biomarkers for the characterization of human MSC using DNA aptamers produced by the SELEX (Systematic Evolution of Ligands by EXponential Enrichment) technique. Our results showed that MSC from different origins bound to DNA candidate aptamers, that is, DNA or RNA oligonucleotides selected from random libraries that bind specifically to biological targets. Aptamer-bound MSC could be isolated by fluorescenceactivated cell sorting (FACS) procedures, enhancing the induction of differentiation into specific phenotypes (chondrocytes, osteocytes and adipocytes) when compared to the whole MSC population. Flow cytometry analyses revealed that candidate aptamers bound to 50% of the MSC population from dental pulp and did not present significant binding rates to human fibroblasts or lymphocytes, both used as negative control. Moreover, immunofluorescence images and confocal analyses revealed staining of MSC by aptamers localized in the surfacemembrane of these cells. The results also showed internal staining of human monocytes by our investigated aptamers. A non-specific control aptamer (CNTR APT) obtained from the random pool was then utilized to compare the specificity of the aptamers bound to the analyzed non-apoptotic cells, showing no staining for MSC. However, 40% of the monocytes bound to the CNTR APT. Normalized data based on the cells bound to candidate aptamers compared to those bound to the CNTR APT, revealed a 10 to 16-fold higher binding rate for MSC against 2-fold for monocytes. Despite its low specificity, monocyte-aptamer binding occurs probably due to the expression of shared markers with MSC, since monocytes are derived from hematopoietic stem cells and are important for the immune system ability to internalize/phagocyte external molecules. Given that, we performed a pull-down assay followed by mass spectrometry analysis to detect which MSC-specific protein or other target epitope not coexpressed by monocytes or the CNTR APT would bind to the candidate aptamer. Distinguishing between MSC and monocyte epitopes is important, as both cells are involved in immunomodulatory effects after MSC transplantations. ADAM17 was found to be a target of the APT10, emerging as a possible biomarker of MSC, since its involvement in the inhibition of the TGF signaling cascade, which is responsible for the differentiation of MSC. Thus, MSC with a higher stemness profile should overexpress the protein ADAM17, which presents a catalytic site with affinity to APT10. Another target of Apt 10 is VAMP3, belonging to a transmembrane protein complex that is involved in endocytosis and exocytosis processes during immune and inflammatory responses. Overall, proteins identified as targets of APT10 may be cell surface MSC biomarkers, with importance for MSC-based cell and immune therapies

Células tronco são células indiferenciadas que podem ser distinguidas de outros tipos celulares por meio da habilidade de se auto renovarem e de se diferenciarem em novos tipos celulares. Células tronco mesenquimais (MSC) são células tronco adultas encontradas em diferentes tecidos como tecido adiposo, polpa de dente e cordão umbilical. Estas células podem se autodividir em células idênticas ou se diferenciarem em células de origem mesodermal. Estas células têm sido estudadas em novas aplicações que envolvem terapia regenerativas. Embora resultados encorajadores tenham sido demonstrados, terapias que utilizam MSC ainda encontram uma grande barreira: a dificuldade no isolamento destas células a partir de um ambiente heterogêneo. MSC são caracterizadas por populações positivas em ensaios de imunomarcação para os epítopos membranares CD29, CD73, CD90 e CD105, presentes também em outros tipos celulares. Assim, o presente trabalho tem o objetivo de identificar novos biomarcadores de MSC de origem humana, utilizando aptâmeros de DNA produzidos pela técnica SELEX (Systematic Evolution of Ligands by EXponential Enrichment) como ferramenta. Nossos resultados mostraram que MSC de diferentes origens ligam-se a aptâmeros (oligonucleotídeos de DNA ou RNA que atuam como ligantes específicos de alvos moleculares) de DNA candidatos que atuam no isolamento de MSC por meio da técnica FACS de separação celular, promovendo uma maior indução de diferenciação em células específicas (condrócitos, osteócitos e adipócitos) comparada com a população total de MSC. Análises de citometria de fluxo mostraram que os aptâmeros candidatos se ligam a 50% das MSC de polpa de dente e não apresentam taxa de ligação significante para fibroblastos e linfócitos de origem humana - utilizados como controles negativo. Além domais, imagens de imunofluorescência e confocal mostraram ligação na superfície da membrana de MSC e a marcação interna de monócitos a estes aptâmeros. Portanto, um aptâmero controle (CNTR APT) foi utilizado para comparar a especificidade dos aptâmeros ligados a células viáveis, mostrando a não ligação deste aptâmero a MSC. Porém, 40% da população de monócitos ligou-se ao CNTR APT. Uma normalização baseada na comparação entre as taxas de ligação entre células ligadas com aptâmeros candidatos e o aptâmero controle gerou uma taxa de especificidade entre 10-16 vezes maior para MSC contra 2,5 vezes para os monócitos. Deste modo, embora os resultados tenham mostrado uma taxa de ligação entre monócitos e aptâmeros, as MSC ligadas aos aptâmeros candidatos possuem uma maior taxa de especificidade devido a uma maior presença de antígenos que são expressos em ambas as células. Um ensaio de Pull Down seguido de espectrometria de massas foi utilizado para a identificação de biomarcadores que se ligariam aos aptâmeros candidatos, e que não seriam co-expressos por monócitos e por antígenos ligados ao aptâmero controle. Deste modo, a proteína ADAM17 foi identificada nas amostras de APT10 ligadas às MSC. Tal proteína está relacionada à inibição de uma cascata de sinalização da família de proteínas TGF, responsável pela diferenciação de MSC. Assim, MSC com maior potencial tronco deveriam expressar ADAM17 em maior quantidade. Tal proteína apresenta um sítio catalítico que demonstra interagir com o APT10, de acordo com predição Docking entre proteína e DNA. Foi identificada também, a proteína VAMP3, que pertence a um complexo proteico transmembranar responsável pelos processos de endocitose e exocitose, e que podem ter um papel importante na liberação de citocinas e outras moléculas relacionadas às respostas imune e inflamatórias. Deste modo, o APT10 identificou proteínas importantes que devem estar relacionas com a melhora de imunoterapias que utilizam MSC

The effect of fat mass and obesity associated proteins mediated mRNA m6A modification on animal fat deposition and its application prospects / 生物工程学报

In the process of animal fat deposition, the proliferation and differentiation of pre-adipocytes and the change of lipid droplet content in adipocytes are regulated by a series of transcription factors and signal pathways. Although researchers have conducted in-depth studies on the transcriptional regulation mechanisms of adipogenesis, there are relatively few reports on post-transcriptional modification on mRNA levels. The modification of mRNA m6A regulated by methyltransferase, demethylase and methylation reading protein is a dynamic and reversible process, which is closely related to fat deposition in animals. Fat mass and obesity associated proteins (FTO) act as RNA demethylases that affect the expression of modified genes and play a key role in fat deposition. This article summarized the mechanism of FTO-mediated demethylation of mRNA m6A in the process of animal fat deposition, suggesting that FTO may become a target for effective treatment of obesity. Moreover, this review summarized the development of FTO inhibitors in recent years.

C/EBPb converts bovine fibroblasts to adipocytes without hormone cocktail induction

BACKGROUND: Adipogenesis and fibrogenesis can be considered as a competitive process in muscle, which may affect the intramuscular fat deposition. The CCAAT/enhancer-binding protein beta (C/EBPb) plays an important role in adipogenesis, which is well-characterized in mice, but little known in bovine so far. RESULTS: In this study, real-time qPCR revealed that the level of C/EBPb was increased during the developmental stages of bovine and adipogenesis process of preadipocytes. Overexpression of C/EBPb promoted bovine fibroblast proliferation through mitotic clonal expansion (MCE), a necessary process for initiating adipogenesis, by significantly downregulating levels of p21 and p27 (p < 0.01). Also, the PPARc expression was inhibited during the MCE stage (p < 0.01). 31.28% of transfected fibroblasts adopted lipid-laden adipocyte morphology after 8 d. Real-time qPCR showed that C/EBPb activated the transcription of early stage adipogenesis markers C/EBPa and PPARc. Expression of ACCa, FASN, FABP4 and LPL was also significantly upregulated, while the expression of LEPR was weakened. CONCLUSIONS: It was concluded C/EBPb can convert bovine fibroblasts into adipocytes without hormone induction by initiating the MCE process and promoting adipogenic genes expression, which may provide new insights into the potential functions of C/EBPb in regulating intramuscular fat deposition in beef cattle.

Myostatin coordinating the proliferation and differentiation of adipose and skeletal muscle cells and energy metabolism balance based on gene chip technology / A miostatina na coordenação da proliferação e diferenciação de células musculares adiposas e esqueléticas e no equilíbrio do metabolismo energético com base na tecnologia de chips genéticos / Msrn, basado en la tecnología de chips genéticos, coordina la fertilización y el metabolismo de las células musculares grasas y óseas

ABSTRACT Myoblasts fuse into multinucleated muscle fibers to form and promote the growth of skeletal muscle. In order to analyze the role of myostatin (MSTN) in body fat, skeletal muscle cell proliferation and differentiation and energy metabolism, this study will use the antisense RNA technology of gene chip technology to study it. The results showed that the MSTN gene regulated the growth and proliferation of myoblasts and affected the development of skeletal muscle by affecting the expression of Cdc42, bnip2, p38 and other genes; knockout or overexpression of the MSTN gene would lead to a trend of fat-related genes from fat synthesis to fat decomposition; after the MSTN gene was knocked down, the expression levels of cpti-b, PPARG and other genes in the cells were corresponding after MSTN overexpression, the relative expression of the PPARG gene decreased. It is suggested that the knockout or overexpression of MSTN may affect lipid accumulation, and cpti-b and PPARG may directly regulate lipid level. It is hoped that this experiment can provide a reference for the study of MSTN effect on fat deposition.

RESUMO Os mioblastos se fundem eM fibras musculares multinucleadas para formar e promover o crescimento do músculo esquelético. A fim de analisar o papel da miostatina (MSTN) na gordura corporal, proliferação de células musculares esqueléticas e diferenciação e metabolismo energético, este estudo utilizará a tecnologia anti-RNA de chips genéticos para estudá-la. Os resultados mostraram que o gene MSTN regulava o crescimento e a proliferação de mioblastos e afetava o desenvolvimento do músculo esquelético, afetando a expressão de Cdc42, bnip2, p38 e outros genes; a eliminação ou sobrexpressão do gene MSTN conduziria a uma tendência de os genes adiposos sintetizarem a gordura até sua decomposição; após a eliminação do gene MSTN, os níveis de expressão de cpti-b, PPARG e outros genes nas células mostraram-se correspondentes após a sobrexpressão do gene MSTN, e a expressão relativa do gene PPARG diminuiu. Sugere-se que a eliminação ou sobrexpressão da MSTN possa afetar a acumulação de lipídeos, e o cpti-b e o PPARG podem regular diretamente o nível lipídico. Espera-se que esta experiência possa fornecer uma referência para o estudo do efeito da MSTN sobre a deposição de gordura.

RESUMEN Los mioblastos se funden en fibras musculares multinucleadas para formar y promover el crecimiento del músculo esquelético. A fin de analizar el papel de la miostatina (MSTN) en la grasa corporal, proliferación de células musculares esqueléticas y diferenciación y metabolismo energético, este estudio utilizará la tecnología anti-RNA de chips genéticos para estudiarla. Los resultados mostraron que el gen MSTN regulaba el crecimiento y la proliferación de mioblastos y afectaba el desarrollo del músculo esquelético, afectando la expresión de Cdc42, bnip2, p38 y otros genes; la eliminación o sobreexpresión del gen MSTN conduciría a una tendencia de que los genes adiposos sinteticen la grasa hasta su descomposición; después de la eliminación del gen MSTN, los niveles de expresión de cpti-b, PPARG y otros genes en las células se mostraron correspondientes después de la sobreexpresión del gen MSTN, y la expresión relativa del gen PPARG disminuyó. Se sugiere que la eliminación o sobreexpresión de la MSTN pueda afectar la acumulación de lipídos, y el cpti-b y el PPARG pueden regular directamente el nivel lipídico. Se espera que esta experiencia pueda proveer una referencia para el estudio del efecto de la MSTN sobre el depósito de grasa.

Alcance de las células madre derivadas de tejido adiposo / Scope of stem cells derived from adipose tissue

Introducción: En el tejido adiposo se han identificado células madre mesenquimales con capacidad autorrenovadora y multipotencial. Mediante digestión enzimática y centrifugado del lipoaspirado se libera una población heterogénea de células denominada fracción vascular estromal, con innumerables potencialidades terapéuticas en el campo de la medicina regenerativa. Objetivo: Actualizar el alcance de las células madre derivadas de tejido adiposo en la terapia regenerativa. Método: Se revisaron 38 artículos entre los años 2000 y 2019 en las bases de datos Scielo, ScienceDirect, Medline y Pubmed. Desarrollo: Las células de la fracción vascular estromal se caracterizan por su capacidad de generar tejido adiposo y vasos sanguíneos, y por la producción de factores de crecimiento que ayudan en la supervivencia de los adipocitos y la formación de una red vascular. El principal mecanismo de acción de las células madre derivadas de tejido adiposo parece deberse a su acción paracrina y a la sinergia con células endoteliales. En el campo de la medicina regenerativa se han utilizado en el tratamiento de cicatrices patológicas y de fibrosis deformantes con impotencia funcional, en las reconstrucciones de secuelas por cáncer y en el cierre precoz de zonas cruentas. Conclusiones: La lipotransferencia es un procedimiento con un mínimo de complicaciones que constituye una de las opciones terapéuticas más empleadas para corregir defectos en los tejidos, debido a que no solo es un medio de relleno, sino que también permite la regeneración y restauración tisular. La presencia de células madre en el tejido adiposo, unido a su accesibilidad, disponibilidad e histocompatibilidad, ha motivado su aplicación cada vez más expandida en la medicina estética, reconstructiva y regenerativa(AU)

Introduction: Mesenchymal stem cells with self-renewing and multipotential capacity have been identified in adipose tissue. By means of enzymatic digestion and centrifugation of the lipoaspirate a heterogeneous population of cells called vascular stromal fraction is released. It has innumerable therapeutic potentialities in the field of regenerative medicine. Objective: To update the scope of stem cells derived from adipose tissue in regenerative therapy. Method: 38 articles published between 2000 and 2019 in the Scielo, ScienceDirect, Medline and Pubmed databases were reviewed. Development: The cells of the vascular stromal fraction are characterized by generating adipose tissue and blood vessels and by the production of growth factors that help in the survival of adipocytes and the formation of a vascular network. The main mechanism of action of stem cells derived from adipose tissue appears to be due to their paracrine action and synergy with endothelial cells. Stem cells derived from adipose tissue have been used in regenerative medicine for the treatment of pathological scars and deforming fibrosis with functional impotence, in the reconstruction of cancer sequelae and in the early closure of bloody areas. Conclusions: Lipotransfer is a procedure with a minimum of complications that constitutes one of the most widely used therapeutic options to correct tissue defects, since it is not only a filling medium, but also allows tissue regeneration and restoration. The presence of stem cells in adipose tissue, together with their accessibility, availability and histocompatibility, has motivated their increasingly widespread application in aesthetic, reconstructive and regenerative medicine(AU)

Inhibition of preadipocyte differentiation by Lycium barbarum polysaccharide treatment in 3T3-L1 cultures

BACKGROUND: Lycium barbarum (also called wolfberry), a famous Chinese traditional medicine and food ingredient, is well recognized for its significant role in preventing obesity; however, the molecular mechanisms underlying its preventive effects on fat accumulation are not well understood yet. The aim of this study was to determine the effects and mechanism of Lycium barbarum polysaccharides (LBP) on the proliferation and differentiation of 3T3-L1 preadipocytes. MTT was used to detect the proliferation of 3T3-Ll preadipocytes. Oil red O staining and colorimetric analysis were used to detect cytosolic lipid accumulation during 3T3-L1 preadipocyte differentiation. Real-time fluorescent quantitative PCR (qPCR) technology was used to detect peroxisome proliferator-activated receptor c (PPARc), CCAAT/enhancer-binding protein a (C/EBPa), adipocyte fatty-acid-binding protein (aP2), fatty acid synthase (FAS), and lipoprotein lipase (LPL) expression. RESULTS: The concentration of LBP from 25 to 200 lg/mL showed a tendency to inhibit the growth of preadipocytes at 24 h, and it inhibited the differentiation of 3T3-L1 preadipocytes in a dose-dependent manner. In the preadipocytes treated with 200 lg/mL LBP, there were reduced lipid droplets in the cytoplasm, and its effect was opposite to that of rosiglitazone (ROS), which significantly reduced the PPARc, C/EBPa, aP2, FAS, and LPL mRNA expression of adipocytes. CONCLUSIONS: LBP exerts inhibitive effects on the proliferation and differentiation of 3T3-L1 preadipocytes and decreases the cytoplasm accumulation of lipid droplets during induced differentiation of preadipocytes toward mature cells. Above phenomenon might link to lowered expression of PPARc, C/EBPa, aP2, FAS, and LPL after LBP treatment. Thus, LBP could serve as a potential plant extract to treat human obesity or improve farm animal carcass quality via adjusting lipid metabolism.

Effects of oleic (18: 1n-9) and palmitic (16: 0) fatty acids on the metabolic state of adipocytes / Efectos de los ácidos grasos oleico (18: 1n-9) y palmítico (16: 0) en el estado metabólico del adipocito

Abstract Background: Elevated serum-free fatty acid (FFA) levels induce insulin resistance (IR) or a protective mechanism to IR development in humans; it depends on FFA type. Objetive: This study explores the effects of oleic (OLA - unsatured) and palmitic (PAM - saturated) fatty acids on insulin action in mature adipocytes effect. Methods: Cells were incubated 18 h with or without OLA and PAM at 250 μM, and 500 μM. After the culture period, were measured: adipocyte viability, size, fatty acids mobilisation, insulin signalling proteins, and glucose uptake. Results: Adipocytes exhibited optimal viability tolerances regardless of the kinds of fatty acids used for treatment. However, adipocytes were hypertrophic after OLA and PAM stimuli. Additionally, lipogenesis (lipid synthesis), and lipolysis (lipid breakdown) were significantly increased by treatment with OLA, or PAM (500 μM) compared to control. Moreover, OLA results showed that there was no significant reduction in signalling cascades, except for a downstream proinflammatory response. Instead, PAM hypertrophic adipocytes were insulin resistant with alteration of proinflammatory and stress markers. Conclusions: Current findings suggest that PAM induces insulin resistance, mitochondrial and reticulum stress on fat cells compared to those treated with OLA that, protects adipocytes to all those alterations.

Resumen Introducción: Los niveles elevados de ácidos grasos libres (AGL) en suero inducen resistencia a insulina (RI) o un mecanismo de protección del desarrollo de RI en humanos, esto depende del tipo de AGL. Objetivo: Este estudio explora los efectos de los ácidos grasos oleico (insaturados - OLA) y palmítico (saturados - PAM) sobre la insulina en adipocitos maduros. Métodos: Las células se incubaron 18 h con o sin OLA y PAM a 250 μM y 500 μM. Después del período de cultivo, se evaluó en adipocitos: viabilidad, tamaño, movilización de ácidos grasos, proteínas de señalización de insulina y absorción de glucosa. Resultados: Los adipocitos mostraron viabilidad óptima independientemente de los tipos de ácidos grasos utilizados en el tratamiento. Los adipocitos eran hipertróficos tras estimulo con OLA y PAM. La lipogénesis (síntesis de lípidos) y la lipólisis (degradación de lípidos) aumentaron significativamente con el tratamiento con OLA o PAM (500 μM) en comparación con el control. En los resultados de OLA no se evidenció una reducción significativa en las cascadas de señalización de insulina, a excepción de una respuesta proinflamatoria posterior. En cambio, los adipocitos hipertróficos tratados con PAM presentaron resistencia a la insulina y alteración de los marcadores proinflamatorios y de estrés. Conclusiones: Nuestros hallazgos sugieren que PAM induce resistencia a la insulina, estrés mitocondrial y del retículo en las células grasas en comparación con aquellos tratados con OLA, AGL que, en cambio, protegen a los adipocitos de todas esas alteraciones.

An in vitro model mimicking the complement system to favor directed phagocytosis of unwanted cells

BACKGROUND: Opsonization, is the molecular mechanism by which target molecules promote interactions with phagocyte cell surface receptors to remove unwanted cells by induced phagocytosis. We designed an in vitro system to demonstrate that this procedure could be driven to eliminate adipocytes, using peptides mimicking regions of the complement protein C3b to promote opsonization and enhance phagocytosis. Two cell lines were used: (1) THP-1 monocytes differentiated to macrophages, expressing the C3b opsonin receptor CR1 in charge of the removal of unwanted coated complexes; (2) 3T3-L1 fibroblasts differentiated to adipocytes, expressing AQP7, to evaluate the potential of peptides to stimulate opsonization. (3) A co-culture of the two cell lines to demonstrate that phagocytosis could be driven to cell withdrawal with high efficiency and specificity. RESULTS: An array of peptides were designed and chemically synthesized p3691 and p3931 joined bound to the CR1 receptor activating phagocytosis (p < 0.033) while p3727 joined the AQP7 protein (p < 0.001) suggesting that opsonization of adipocytes could occur. In the co-culture system p3980 and p3981 increased lipid uptake to 91.2% and 89.0%, respectively, as an indicator of potential adipocyte phagocytosis. CONCLUSIONS: This in vitro model could help understand the receptor­ligand interaction in the withdrawal of unwanted macromolecules in vivo. The adipocyte-phagocytosis discussed may help to control obesity, since peptides of C3b stimulated the CR1 receptor, promoting opsonisation and phagocytosis of lipidcontaining structures, and recognition of AQP7 in the differentiated adipocytes, favored the phagocytic activity of macrophages, robustly supported by the co-culture strategy.

The influence of adipocyte-derived stem cells (ASCs) on the ischemic epigastric flap survival in diabetic rats

ABSTRACT Purpose: To assess the effects of adipocyte-derived stem cell (ASC)-injection on the survival of surgical flaps under ischemia in diabetic rats. Methods: Diabetes was induced in 30 male Wistar rats using streptozotocin (55 mg/kg). After eight weeks, epigastric flap (EF) surgery was performed. The animals were divided into control (CG), medium-solution (MG), and ASC groups. The outcomes were: the survival area (SA), the survival/total area rate (S/TR), and expression levels (EL) of genes: C5ar1, Icam1, Nos2, Vegf-a. Results: In the ASC group, compared to CG, we observed improved flap SA (CG-420 mm2 vs. ASC-720 mm2; p=0.003) was observed. The S/TR analysis was larger in the ASC group (78%) than the CG (45%). This study showed an increase in the Vegf-a EL in the ASC group (2.3) vs. CG (0.93, p=0.0008). The Nos2 EL increased four-fold in the ASC group compared to CG, and C5ar1 EL decreased almost two-fold in the ASC group vs. the CG (p=0.02). There was no difference among the groups regarding Icam1 EL. Compared to the MG, the ASC group had a bigger flap SA (720 mm2 vs. 301 mm2, respectively), a bigger S/TR (78% vs. 32%, p=0.06, respectively) and increased EL of Vegf-a (2.3 vs. 1.3, respectively). No difference between ASC-group and MG was seen regarding Nos2 (p=0.08) and C5ar1 (p=0.05). Conclusions: This study suggests that ASCs increase the survival of EF under IR in diabetic rats.

Lipoma Arborescente: uma causa de sinovite crônica? / Lipoma Arborescens: a cause of chronic synovitis?

O lipoma arborescente é uma causa incomum de lesão intra-articular que se apresenta como aumento de volume articular indolor, lentamente progressivo, que persiste por muitos anos e é acompanhado por derrames articulares intermitentes. O envolvimento de sítios extra-articulares é incomum, mas pode ocorrer em bainhas tendíneas e bursas. A ressonância magnética é o melhor exame para o diagnóstico, embora a biópsia sinovial possa ser necessária em alguns casos. Relatamos três casos com o objetivo de destacar o espectro clínico da doença, as características da imagem e a resposta ao tratamento imunossupressor.

Lipoma arborescens is an uncommon cause of intra-articular masses that presents as slowly progressive painless swelling of the joint, which persists for many years and is accompanied by intermittent effusions. Extra-articular site(s) involvement is unusual, but can occur in tendon sheaths and bursas. Magnetic resonance imaging is the best diagnostic exam, although synovial biopsy may be necessary. We report three cases in order to highlight the clinical spectrum and imaging features of the disease, so that early diagnosis and appropriate treatment can be given.

The molecular mechanism of fibroblast growth factor 21-inhibited leptin expression in adipocytes / 生理学报

The present study was aimed to clarify the signaling molecular mechanism by which fibroblast growth factor 21 (FGF21) regulates leptin gene expression in adipocytes. Differentiated 3T3-F442A adipocytes were used as study object. The mRNA expression level of leptin was detected by fluorescence quantitative RT-PCR. The phosphorylation levels of proteins of signal transduction pathways were detected by Western blot. The results showed that FGF21 significantly down-regulated the mRNA expression level of leptin in adipocytes, and FGF21 receptor inhibitor BGJ-398 could completely block this effect. FGF21 up-regulated the phosphorylation levels of ERK1/2 and AMPK in adipocytes. Either ERK1/2 inhibitor SCH772984 or AMPK inhibitor Compound C could partially block the inhibitory effect of FGF21, and the combined application of these two inhibitors completely blocked the effect of FGF21. Neither PI3K inhibitor LY294002 nor Akt inhibitor AZD5363 affected the inhibitory effect of FGF21 on leptin gene expression. These results suggest that FGF21 may inhibit leptin gene expression by activating ERK1/2 and AMPK signaling pathways in adipocytes.

CRISPR/Cas9 knockout plin1 enhances lipolysis in 3T3-L1 adipocytes / 生物工程学报

We used CRISPR/Cas9 to delete plin1 of 3T3-L1 preadipocyte, to observe its effect on lipolysis in adipocytes and to explore regulatory pathways. We cultured 3T3-L1 preadipocytes, and the plin1 knockout vectors were transfected by electroporation. Puromycin culture was used to screen successfully transfected adipocytes, and survival rates were observed after transfection. The optimized "cocktail" method was used to differentiate 3T3-L1 preadipocytes. The glycerol and triglyceride contents were determined by enzymatic methods. The changes in lipid droplet form and size were observed by Oil red O staining. The protein expression of PLIN1, PPARγ, Fsp27, and lipases was measured by Western blotting. RT-PCR was used to measure the expression of PLIN1 and lipases mRNA. After the adipocytes in the control group were induced to differentiate, the quantity of tiny lipid droplets was decreased, and the quantity of unilocular lipid droplets was increased and arranged in a circle around the nucleus. Compared with the control group, the volume of unilocular lipid droplets decreased, and the quantity of tiny lipid droplets increased after induction of adipocytes in the knockout group. The expression of PLIN1 mRNA and protein in the adipocytes was significantly inhibited (P<0.05); glycerol levels increased significantly (0.098 4±0.007 6), TG levels decreased significantly (0.031 0±0.005 3); mRNA and protein expression of HSL and ATGL increased (P<0.05); PPARγ and Fsp27 expression unchanged in adipocytes. The above results indicate that the knockout of plin1 enhances the lipolysis of 3T3-L1 adipocytes by exposing lipids in lipid droplets and up-regulating lipases effects.

Metabolic Crosstalk between the Heart and Fat

It is now recognized that the heart can behave as a true endocrine organ, which can modulate the function of other tissues. Emerging evidence has shown that visceral fat is one such distant organ the heart communicates with. In fact, it appears that bi-directional crosstalk between adipose tissue and the myocardium is crucial to maintenance of normal function in both organs. In particular, factors secreted from the heart are now known to influence the metabolic activity of adipose tissue and other organs, as well as modulate the release of metabolic substrates and signaling molecules from the periphery. This review summarizes current knowledge regarding primary cardiokines and adipokines involved in heart-fat crosstalk, as well as implications of their dysregulation for cardiovascular health.

Monitorização de células-tronco mesenquimais injetadas via retrobulbar próximas ao nervo óptico lesados de coelhos / Monitoring of stem cells from adipose tissue injected via retrobulbar next to previously injured optic nerve of rabbits

Resumo Objetivo: Verificar a presença das células-tronco mesenquimais (MSC) na área próxima ao nervo óptico de coelhos previamente lesado com álcool absoluto. Métodos: Os 12 coelhos da raça Nova Zelândia foram distribuídos em 2 lotes. Após sedação, cada olho do animal recebeu uma injeção retrobulbar de 1 ml de álcool absoluto em um dos olhos e de 1 ml de solução fisiológica 0,9% (SF) no olho contralateral. Após 15 dias deste procedimento inicial todos os olhos dos animais pertencentes ao lote A, receberam via retrobulbar, uma solução contendo MSC de tecido adiposo humano e previamente marcadas com Qdots,. Todos os olhos dos animais do lote B receberam solução PBS. Resultados: Após 15 dias desta última aplicação os animais foram sacrificados e as lâminas foram analisadas. A presença das MSC foi observada em 100% dos olhos dos animais do lote A. Conclusão: Os resultados sugerem que a marcação prévia das MSC com Qdots permitiu o acompanhamento das mesmas na região aplicada e em áreas mais internas do nervo óptico. A permanência de MSC após 15 dias de aplicação ao redor do nervo óptico sugere a viabilidade e possível participação das mesmas no processo de regeneração do tecido lesado. Nas condições deste estudo, a via de aplicação retrobulbar permitiu a mobilização das células tronco do local de aplicação até áreas centrais dos nervos ópticos nos animais do lote A, sugerindo que esta poderá ser uma via de acesso eficaz para as MSC no processo de regeneração de neuropatias ópticas.

Abstract Obtective: To verify the presence of mesenchymal stem cells (MSC) in the area close to the optic nerve of previously injured with absolute alcohol. Methods: Twelve New Zealand breed rabbits were divided into two groups, and after sedation, each eye of the animal received a retrobulbar injection of 1 ml of absolute ethanol in one eye, and 1 ml of physiological solution 0.9 % (PS) in the contralateral eye. After 15 days all eyes of animals belonging to group A, received via retrobulbar a solution containing MSCs from human adipose tissue (AT) and previously marked with Qdots, while all eyes of animals from group B received solution containing PBS. Results: The presence of MSC was observed in 100% of the eyes of the animals of group A and the more central areas near and into the optic nerve. Conclusion: The results suggest that the appointment of MSC with Qdots allowed their follow-up applied in the region and in the inner areas of the optic nerve. The MSC permanence after 15 days of application around the optic nerve suggests the feasibility and possible involvement of the same during the damaged tissue regeneration process. Under the conditions of this study, the route of retrobulbar application and the presence of the stem cells to the central areas of the optic nerves in animals of group A, suggests that this might be an effective approach for MSCs in regeneration process of optic neuropathies.