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Acta Physiologica Sinica ; (6): 426-432, 2020.
Article in Chinese | WPRIM | ID: wpr-827045


The purpose of the present study was to investigate the effects of forkhead box O4 (FOXO4) on the senescence of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs). The hUC-MSCs were induced to senescence by natural passage, and FOXO4 expression was inhibited by lentiviral shRNA transfection. The hallmark of cell senescence was analyzed by β-galactosidase staining, and the cell viability was assayed by CCK-8 method. Flow cytometry was used to investigate the apoptosis of hUC-MSCs. The expression levels of Bcl-2, Bax, FOXO4, interleukin 6 (IL-6) and cleaved Caspase-3 were detected by qPCR and Western blot. Immunofluorescence staining was used to detect FOXO4 expression. The amount of IL-6 secreted by hUC-MSCs was detected by ELISA. The results showed that, compared with the passage 1, senescent hUC-MSCs showed up-regulated expression levels of Bax and FOXO4, down-regulated expression levels of Bcl-2 and cleaved Caspase-3, and increased IL-6 mRNA expression and secretion. FOXO4 inhibition in senescent hUC-MSCs promoted cell apoptosis, reduced cell viability, and inhibited the mRNA expression and secretion of IL-6. These results suggest that FOXO4 maintains viability and function of senescent hUC-MSCs by repressing their apoptosis response, thus accelerating senescence of the whole cell colony.

Humans , Apoptosis , Cell Cycle Proteins , Cell Survival , Cellular Senescence , Forkhead Transcription Factors , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells , Transcription Factors , Umbilical Cord
Article in Chinese | WPRIM | ID: wpr-360042


<p><b>OBJECTIVE</b>To study the regulation of SIRT1 by transcription factor SREBP-1 in adipogeneic differentiation of bone marrow mesenchymal stem cells (BMMSC).</p><p><b>METHODS</b>Oil red O staining was used to identify the adipogenic differentiation of BMMSC; the mRNA transcription levels of AP2, LPL, SREBF-1, SIRT1 gene were detected by RT-PCR; the expession level of SREBP-1 was determined by Western-blot. The chromatin immunoprecipitation (ChIP) assay was used to investigate the binding of SREBP-1 to SIRT1 promoter.</p><p><b>RESULTS</b>BMMSC exposed to adipogenesis inducing medium become mature adipocytes at day 14; the mRNA transcription levels of AP2, LPL, SREBF-1, SIRT1 genes were up-regulated in adipocyte differentiation of BMMSC; the protein level of SREBP-1 was higher obviously; SIRT1 gene sequences was succesfully amplified from the genomic DNA immunoprecipitated by SREBP-1 antibody.</p><p><b>CONCLUSION</b>SREBP-1 can bind to the promoter region of the SIRT1 gene in adipogenesis of BMMSC, and may be involved in the transcriptional regulation of the SIRT1 gene.</p>

Humans , Adipocytes , Cell Biology , Adipogenesis , Cell Differentiation , Cells, Cultured , Chromatin Immunoprecipitation , Gene Expression Regulation , Mesenchymal Stem Cells , Cell Biology , Promoter Regions, Genetic , Sirtuin 1 , Metabolism , Sterol Regulatory Element Binding Protein 1 , Metabolism , Up-Regulation
Article in Chinese | WPRIM | ID: wpr-272481


<p><b>OBJECTIVE</b>To study the effects of LIF combined with bFGF on the proliferation, stemness and senescence of hUC-MSC.</p><p><b>METHODS</b>Experiments were divided into 4 groups: control group, in which the cells were treated with complete medium (α-MEM containing 10% FBS); group LIF, in which the cells were treated with complete medium containing 10 ng/ml LIF; group bFGF, in which the cells were treated with complete medium containing 10 ng/ml bFGF; combination group, in which the cells were treated with complete medium containing 10 ng/ml LIF and 10 ng/ml bFGF. The growth curves of hUC-MSC at passage 4 in different groups were assayed by cell counting kit 8. Cellular morphologic changes were observed under inverted phase contrast microscope; hUC-MSC senescence in different groups was detected by β-galactosidase staining. The expression of PCNA, P16, P21, P53, OCT4 and NANOG genes was detected by RT-PCR.</p><p><b>RESULTS</b>The cell growth curves of each group were similar to the S-shape; the cell proliferation rate from high to low as follows: that in the combination group > group bFGF > group LIF > control group. Senescence and declining of proliferation were observed at hUC-MSC very early in control group; the cells in group LIF maintained good cellular morphology at early stage, but cell proliferation was slow and late senescence was observed; a few cells in group bFGF presented signs of senescence, but with quick proliferation; the cells in combination group grew quickly and maintained cellular morphology of hUC-MSC for long time. The LIF and bFGF up-regulated the expression of PCNA, OCT4 and NANOG, while they down-regulated the expression of P16, P21, P53, and their combinative effects were more significant.</p><p><b>CONCLUSION</b>LIF combined with bFGF not only can promote the proliferation and maintenance of stemness of hUC-MSC, but also can delay the senescence of hUC-MSC.</p>

Humans , Cell Cycle , Cell Differentiation , Cell Proliferation , Cells, Cultured , Cyclin-Dependent Kinase Inhibitor p16 , Metabolism , Cyclin-Dependent Kinase Inhibitor p21 , Metabolism , Fibroblast Growth Factor 2 , Pharmacology , Genes, Homeobox , Leukemia Inhibitory Factor , Pharmacology , Mesenchymal Stem Cells , Cell Biology , Octamer Transcription Factor-3 , Metabolism , Organic Chemicals , Proliferating Cell Nuclear Antigen , Metabolism , Tumor Suppressor Protein p53 , Metabolism , Umbilical Cord , Cell Biology