RESUMO
Reference gene selection in mouse oocytes is an important task required to perform further adequate analysis of target gene expression levels. In the current work we have analyzed expression stability of the seven most commonly used reference genes (Actb, Eef1e1, Gapdh, H2afz, Ppia, Rpl4 and Ubc) in mouse oocytes at the germinal vesicle (GV) stage. We have performed analysis of expression stability of the above-mentioned reference genes with the three most commonly used software tools: geNorm, BestKeeper and NormFinder. Taking into account the results obtained from all of these programmes Gapdh, Rpl4 and H2afz seem to be suitable candidate reference genes in GV oocytes of mouse.
Assuntos
Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento , Oócitos/metabolismo , Oogênese/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Animais , Feminino , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/genética , Camundongos , Oócitos/citologia , Fatores de Alongamento de Peptídeos/toxicidade , Padrões de Referência , Proteínas Ribossômicas/genética , Proteínas Supressoras de Tumor/toxicidadeRESUMO
PURPOSE: To select reference genes with stable messenger RNA (mRNA) expression for quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) analysis of vitrified/thawed human ovarian tissue and to evaluate in human ovarian tissue the levels of key proteins which are commonly used as reference proteins. METHODS: Pieces of ovarian tissue were obtained during laparoscopy from patients (n = 10, 24-36 years old) who suffered from types of cancer that does not affect reproductive system. Tissue strips from the intact group were immediately placed into liquid nitrogen. Tissue strips from the second group were successively placed into solutions with cryoprotective agents. Then, these strips were rapidly placed into liquid nitrogen. After thawing, ovarian tissue strips were cultured during 2 h in complete growth medium. Gene expression levels were measured using quantitative RT-PCR. Also, protein levels of three key reference genes were measured using Western blot. Statistical analysis of obtained data was performed by BestKeeper, NormFinder, and geNorm software utilities; correlation coefficients were also calculated. RESULTS: The most suitable reference genes for qRT-PCR analysis of human cortical ovarian tissue after cryopreservation by vitrification are genes of ribosomal proteins RPL4, RPLP0, RPS18, and heat shock protein HSP90AB1. The protein levels of three commonly used reference genes (ACTB, GAPDH, and HSP90) were measured in two groups of samples of human ovarian tissue: intact and vitrified/thawed. The levels of ACTB, GAPDH, and HSP90 proteins were similar in native and vitrified/thawed samples. CONCLUSION: Selection of suitable reference genes is the first aim of any research dedicated to the investigation of gene expression, because the interpretation of obtained results largely depends on selection of appropriate reference genes. Nowadays, there are many mathematical approaches allowing to select not only single reference gene but also a group of the most stably expressed reference genes. The use of mathematical models which take into account multiple reference genes will allow to obtain more accurate data on the expression of target genes.
Assuntos
Criopreservação , Regulação da Expressão Gênica no Desenvolvimento/genética , Ovário/metabolismo , Proteínas Ribossômicas/genética , Adulto , Feminino , Perfilação da Expressão Gênica , Proteínas de Choque Térmico HSP90/genética , Humanos , Ovário/crescimento & desenvolvimento , Padrões de Referência , VitrificaçãoRESUMO
One of the essential goals in regenerative medicine is microvascularization which enables an effective blood supply within de novo constructed tissues and organs. In our study, we used two common multipotent mesenchymal stromal cell (MMSC) sources (subcutaneous adipose tissue and Wharton's jelly of the umbilical cord) where is a subpopulation of endothelial precursors. In the medium supplemented with VEGF, the 3D cultures of UC MMSCs and ADSCs promoted the endothelial cell differentiation. To evaluate their ability to form a capillary-like network, we encapsulated spheroids within non-modified and PEGylated fibrin hydrogels. The PEGylated hydrogel supported better the formation of multibranched cords than the pure fibrin gel. Analysis of tubule growth rate, length, and branching showed that the differentiated ADSCs had higher angiogenic potential than the differentiated hUC MMSCs. Our study can be a basis for the development of new strategies in tissue engineering and treatment of vascular diseases.
Assuntos
Adipócitos/citologia , Fibrina/química , Células-Tronco Mesenquimais/citologia , Neovascularização Fisiológica , Células Estromais/citologia , Cordão Umbilical/citologia , Técnicas de Cultura de Células , Diferenciação Celular , Separação Celular , Géis/química , Humanos , Hidrogéis/química , Microscopia de Contraste de Fase , Medicina Regenerativa , Esferoides Celulares , Engenharia Tecidual/métodos , Geleia de Wharton/citologiaRESUMO
It is known that stem and progenitor cells open new possibilities for restoring injured eye tissues. Limbal eye zone, formed mainly by derivatives of neural crest, is the main source of stem cells for regeneration. The current study considers development of innovative technology for obtaining 3D spheroids from L-MMSC. It was shown that under 3D conditions L-MMSC due to compactization and mesenchymal-epithelial transition self-organize into cellular reparative modules. Formed L-MMSC spheroids retain and promote undifferentiated population of stem and progenitor limbal cells, as supported by expression of pluripotency markers - Oct4, Sox2, Nanog. Extracellular matrix synthetized by cells in spheroids allows retaining the functional potential of L-MMSC that are involved in regeneration of both anterior and, probably, posterior eye segment.