RESUMEN
Objective @#To examine the role of LMO4 in the regulation of endothelial cell differentiation and angio- genesis in murine embryonic stem cells (mESC) ．@*Methods @#Mouse Lmo4 cDNA was obtained from MEL cells by using the reverse transcription-polymerase chain reaction (RT-PCR) and subcloned into the expression vector pFG to generate the pFLG ，in which contained Flk-1 promoter to drive Lmo4 expresses in only FLK-1 + cells．The mESC were transfected with pFG or pFLG plasmids and subsequently screened with geneticin ( G418) to produce cell clones． These cell clones were named mESC /pFG and mESC /pFLG ，respectively． The mESC /pFG and mESC /pFLG were cultured in the differentiation medium for either 4 days or 10 days to generate embryoid bodies (EB) ．The 10-day embryoid bodies ( 10 d-EBs) carrying the pFG and pFLG vectors were subsequently stimulated to generate the blast-colony forming cells (BL-CFC) ，which indicated the presence of hemangioblasts．The endo- thelial cell sprouting analysis was performed by using 10 d-EBs．The expression of the interest genes was detected by using qualitative RT-PCR or Western blot analysis. @*Results @#The pFLG expression vector was successfully con- structed through PCR identification．The mESC /pFG and mESC /pFLG cells were obtained after transfected with the pFG or pFLG vectors and selected by G418．The cells spontaneously differentiate to generate EBs，in which some green fluoresce cells were present．Western blot analysis showed that a significant increase in LMO4 expression in both 4 d-EB and 10 d-EB when compared to mESC．BL-CFC analysis showed that the 4 d-EB/ pFLG had a higher cloning efficiency ( 7. 70% ± 1. 27% ) ，comparing with that of the 4 d-EB/ pFG ( 1. 15% ± 0. 48% ) ( P = 0. 021) ．Quantitative RT-PCR results showed that the expression of Flk-1，C-kit，Tie-2 and Ve-cad genes in 10 d- EBs /pFLG increased more than 2-fold compared to 10 d-EBs /pFG．The endothelial cell sprouting analysis result showed a significant increase in the number and length of new blood vessels in 10 d-EB/ pFLG compared to 10 d- EB/ pFG (P＜0. 05) ．@*Conclusion @#Overexpression of LMO4 promotes hemangioblast differentiation from mESC， and benefits for endothelial cell differentiation and angiogenesis.
RESUMEN
Objective: To investigate the overexpression of paired box gene 6(Pax6) gene in mouse embryonic stem cells and obtain cell line, which is the basis for further differentiation of Pax6/mouse embryonic stem cells (mESCs). Methods: mESCs were cultured in vitro, and the recombinant vector pEFla-Pax6-IRES-AcGFP and the empty vector pEFlot-IRES-AcGFP were transfected into mESCs by liposome method respectively. The cells were screened by G418 gradient and fluorescent protein. The expression of Pax6 was detected by RT-PCR, immunofluorescence and Western blotting and the proportion of Pax6/mESCs positive cells was detected by flow cytometry. The obtained cell line was detected by cell immunofluorescence for stem cell markers stage specific embryonic antigen 1 (SSEA1) and octamer binding transcription factor 4(0CT4), and the pluripotency was detected by alkaline phosphatase staining. Pax6/mESCs cells were subcutaneously transplanted, and the grafts were observed by HE staining to observe their differentiation ability. Results: Pax6 was successfully expressed in mESCs. After screening by G418, the cell line Pax6/mESCs was obtained, and the flow rate showed positive rate about 90%. Immunofluorescence showed that stem cell markers SSEA1 and 0CT4 were positively expressed and alkaline phosphatase stained cells were stained brownish black, and transplantation in vivo could differentiate into three germ layers. Conclusion: Pax6 is successfully expressed in mESCs. After screening by G418, the cell line Pax6/mESCs is obtained and shows the good stem cell characteristics.
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In the experimental group (shh inhibited group), there were significant decreases in the expression of Oct4, Nanog, Shh, GATA4, Brachyury and Goosecoid, while increases were observed for TAT and Pdx1. The expression of Sox17 did not differ between two control and experimental groups. In experimental group, the amount of GSC positive cells was somehow lower but it seems that there was no difference for Sox17. Shh inhibition induces ESCs to differentiate toward definitive endoderm by committing mesendodermal lineages.
Asunto(s)
Animales , Diferenciación Celular , Línea Celular , Linaje de la Célula , Cartilla de ADN , Ditizona/farmacología , Células Madre Embrionarias/citología , Endodermo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/metabolismo , Proteínas de Homeodominio/metabolismo , Inmunohistoquímica , Mesodermo/metabolismo , Ratones , Microscopía Fluorescente , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
In order to further study mouse embryonic stem cells(ES cells),lentiviral vector PLL-IRES-Nanog-Neo was constructed.Mouse ES cells overexpressed nanog by mediation of lentiviral were cultured on mouse fetal fibroblast feeders after 2 weeks under G418 media and examined according to gowth characteristics. Results were showed that 918 bp nanog fragments were expressed in mouse ES cells mediated by lentiviral vector PLL-IRES-Nanog-Neo,mouse nanog-ES cells were taken on mass-like image and positve with alkaline phosphatase staining and Oct4 and SSEA1 immunocytochemistry under no LIF condition in the media. It is concluded that mouse ES cells Elevated nanog gene expression by mediation of lentiviral were constucted and cultured.