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1.
Cell Mol Biol Lett ; 24: 14, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30815013

RESUMO

BACKGROUND: The proliferation and adipogenic differentiation of adipose stromal cells (ASCs) are complex processes comprising major phenotypical alterations driven by up- and downregulation of hundreds of genes. Quantitative RT-PCR can be employed to measure relative changes in the expression of a gene of interest. This approach requires constitutively expressed reference genes for normalization to counteract inter-sample variations due to differences in RNA quality and quantity. Thus, a careful validation of quantitative RT-PCR reference genes is needed to accurately measure fluctuations in the expression of genes. Here, we evaluated candidate reference genes applicable for quantitative RT-PCR analysis of gene expression during proliferation and adipogenesis of human ASCs with the immunophenotype DLK1+/CD34+/CD90+/CD105+/CD45-/CD31-. METHODS: We evaluated the applicability of 10 candidate reference genes (GAPDH, TBP, RPS18, EF1A, TFRC, GUSB, PSMD5, CCNA2, LMNA and MRPL19) using NormFinder, geNorm and BestKeeper software. RESULTS: The results indicate that EF1A and MRPL19 are the most reliable reference genes for quantitative RT-PCR analysis of proliferating ASCs. PSMD5 serves as the most reliable endogenous control in adipogenesis. CCNA2 and LMNA were among the least consistent genes. CONCLUSIONS: Applying these findings for future gene expression analyses will help elucidate ASC biology.


Assuntos
Gordura Abdominal/citologia , Perfilação da Expressão Gênica/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Gordura Abdominal/fisiologia , Adipogenia , Proliferação de Células , Perfilação da Expressão Gênica/normas , Humanos , Reação em Cadeia da Polimerase em Tempo Real/normas , Padrões de Referência , Células Estromais/fisiologia
2.
Islets ; 10(6): 221-238, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30521410

RESUMO

Assessing the response of pancreatic islet cells to glucose stimulation is important for understanding ß-cell function. Zebrafish are a promising model for studies of metabolism in general, including stimulus-secretion coupling in the pancreas. We used transgenic zebrafish embryos expressing a genetically-encoded Ca2+ sensor in pancreatic ß-cells to monitor a key step in glucose induced insulin secretion; the elevations of intracellular [Ca2+]i. In vivo and ex vivo analyses of [Ca2+]i demonstrate that ß-cell responsiveness to glucose is well established in late embryogenesis and that embryonic ß-cells also respond to free fatty acid and amino acid challenges. In vivo imaging of whole embryos further shows that indirect glucose administration, for example by yolk injection, results in a slow and asynchronous induction of ß-cell [Ca2+]i responses, while intravenous glucose injections cause immediate and islet-wide synchronized [Ca2+]i fluctuations. Finally, we demonstrate that embryos with disrupted mutation of the CaV1.2 channel gene cacna1c are hyperglycemic and that this phenotype is associated with glucose-independent [Ca2+]i fluctuation in ß-cells. The data reveal a novel central role of cacna1c in ß-cell specific stimulus-secretion coupling in zebrafish and demonstrate that the novel approach we propose - to monitor the [Ca2+]i dynamics in embryonic ß-cells in vivo - will help to expand the understanding of ß-cell physiological functions in healthy and diseased states.


Assuntos
Canais de Cálcio Tipo L/metabolismo , Cálcio/metabolismo , Embrião não Mamífero/metabolismo , Células Secretoras de Insulina/metabolismo , Animais , Animais Geneticamente Modificados , Peixe-Zebra
3.
Stem Cell Res ; 22: 1-12, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28549249

RESUMO

To precisely characterize CD146 in adipose stromal/progenitor cells (ASCs) we sorted the stromal vascular faction (SVF) of human abdominal subcutaneous white adipose tissue (sWAT) according to cell surface (cs) expression of CD146, DLK1 and CD34. This test identified three main SVF cell populations: ~50% cs-DLK1-/cs-CD34+/cs-CD146- ASCs, ~7.5% cs-DLK1+/cs-CD34dim/+/cs-CD146+ and ~7.5% cs-DLK1+/cs-CD34dim/+/cs-CD146- cells. All cells contained intracellular CD146. Whole mount fluorescent IHC staining of small vessels detected CD146+ endothelial cells (CD31+/CD34+/CD146+) and pericytes (CD31-/CD34-/CD146+ ASCs). The cells in the outer adventitial layer showed the typical ASC morphology, were strongly CD34+ and contained low amounts of intracellular CD146 protein (CD31-/CD34+/CD146+). Additionally, we detected wavy CD34-/CD146+ and CD34dim/CD146+ cells. CD34dim/CD146+ cells were slightly more bulky than CD34-/CD146+ cells. Both CD34-/CD146+ and CD34dim/CD146+ cells were detached from the inner pericyte layer and protruded into the outer adventitial layer. Cultured early passage ASCs contained low levels of CD146 mRNA, which was expressed in two different splicing variants, at a relatively high amount of the CD146-long form and at a relatively low amount of the CD146-short form. ASCs contained low levels of CD146 protein, which consisted predominantly long form and a small amount of short form. The CD146 protein was highly stable, and the majority of the protein was localized in the Golgi apparatus. In conclusion, the present study contributes to a better understanding of the spatial localization of CD34+/CD146+ and CD34-/CD146+ cells in the adipose niche of sWAT and identifies CD146 as intracellular protein in cs-DLK1-/cs-CD34+/cs-CD146- ASCs.


Assuntos
Adipócitos/metabolismo , Antígeno CD146/biossíntese , Células Estromais/metabolismo , Adipócitos/citologia , Antígenos CD34/biossíntese , Antígenos CD34/genética , Antígeno CD146/genética , Proteínas de Ligação ao Cálcio , Diferenciação Celular/fisiologia , Células Cultivadas , Citometria de Fluxo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Células Estromais/citologia
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