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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
Basic & Clinical Medicine ; (12): 251-254, 2018.
Article in Chinese | WPRIM | ID: wpr-693880


Exosomes, small vesicles of endocytic origin, have attracted attention in bone regeneration field. The vesicles travel between cells and deliver functional cargoes like proteins and RNAs, thereby regulating targeted cells dif-ferentiation, commitment, function, and proliferation. Exosomes directly regulate MSCs into the osteoblastic lineage, stimulate bone regeneration by directly regulating osteoblast proliferation and activity, regulate osteoclast maturation and activity and stimulate bone growth and regeneration by increasing vessel formation. Meantime, exosomes resolves toxicity and immunogenicity problems caused by biomaterial treatment. Furthermore, compared with living cell trans-plantation, exosomes present a lower risk for severe complication(such as tumors, emboli formation).

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-328251


<p><b>OBJECTIVE</b>To explore the effect of total flavonoids of Herba Epimedium (FHE) on BMP-2/RunX2/OSX signaling pathway in promoting osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).</p><p><b>METHODS</b>Passage 3 BMSCs were randomly divided into the control group, the experimental group, and the inhibitor group. BMSCs in the control group were cultured in 0.2% dimethyl sulfoxide + Osteogenuxic Supplement (OS) fluid + DMEM/F12 culture media. BMSCs in the experimental group were intervened by 20 microg/mL FHE. BMSCs in the inhibitor group were intervened by 20 microg/mL FHE and 1 microg/mL NOGGIN recombinant protein. At day 9 alkaline phosphatase (ALP) activity was measured. Calcium nodules were stained by alizarin red staining and the density was observed. The transcription expression of osteogenic differentiation-related proteins (type I collagen, osteocalcin, and osteopontin) and related factors of BMP-2/RunX2/OSX signaling pathway was assayed by RT-PCR.</p><p><b>RESULTS</b>Compared with the control group, ALP activities were enhanced and the density of calcium nodules significantly increased; type I collagen, osteocalcin, and osteopontin expression levels were increased in the experimental group. The expression of osteogenesis-related transcription factor was also increased in the experimental group. Noggin recombinant protein inhibited FHE promoting BMSCs osteogenesis in the inhibitor group. Compared with the experimental group, ALP activity decreased (P < 0.05), the density of calcium nodules was lowered, expression levels of type I collagen, osteocalcin, osteopontin significantly decreased (P < 0.05) in the inhibitor group.</p><p><b>CONCLUSION</b>20 microg/mL FHE promoted osteogenic differentiation process of BMSCs by BMP-2/RunX2/OSX signaling pathway.</p>

Humans , Bone Morphogenetic Protein 2 , Metabolism , Cell Differentiation , Cells, Cultured , Collagen Type I , Metabolism , Core Binding Factor Alpha 1 Subunit , Metabolism , Drugs, Chinese Herbal , Pharmacology , Epimedium , Chemistry , Flavonoids , Pharmacology , Mesenchymal Stem Cells , Cell Biology , Osteocalcin , Metabolism , Osteogenesis , Osteopontin , Metabolism , Signal Transduction , Sp7 Transcription Factor , Transcription Factors , Metabolism
Article in Chinese | WPRIM | ID: wpr-230094


To investigate the effect of icaritin (ICT) combined with GDF-5 on chondrogenic differentiation of bone marrow stromal cells (BMSCs), and discuss the action of Wnt signaling pathway, full bone marrow adherent method was used to isolate and culture SD rats BMSCs, and the cells at P3 generation were taken and divided into 6 groups: BMSCs group, ICT group, GDF-5 group, GDF-5+ICT group, GDF-5+ICT+SB216763 group, and GDF-5+ICT+ XAV-939 group. The cells were induced and cultured for 14 days. The morphology change was observed by inverted microscope. Alcian blue staining method was used to detect the changes of proteoglycans. RT-PCR was used to detect the mRNA expressions of aggrecan, Col2, Sox9, Dvl1, Gsk3β, and β-catenin. The protein expressions of collagen 2 (COL2) and β-catenin were detected by Western blot. The results indicated that, compared with the BMSCs group, gradual increase was present in proteoglycan Alcian blue staining; mRNA expressions of cartilage differentiation marker genes aggrecan, COL2, Sox9 and the protein expression of COL2, as well as mRNA and protein expressions of Wnt signaling pathway-related gene β-catenin, but with gradual decrease in Gsk3β mRNA expressions in GDF-5 group, GDF-5+ICT group and GDF-5+ICT+SB216763 group. On the contrary, compared with GDF-5+ICT group, there was a decrease in expressions of Dvl1, and β-catenin related to chondrogenic differentiation and Wnt signaling pathway, a increase in Gsk3β mRNA expression, and also a decrease in protein expressions of COL2 and β-catenin in GDF-5+ICT+XAV-939 group, with statistically significant difference between two groups. GDF-5 in combination with icaritin can induce chondrogenic differentiation of BMSCs in rats, and icaritin (ICT) can promote the chondrogenic differentiation. ICT can promote the chondrogenic differentiation of BMSCs in vitro probably by activating the Wnt/β-catenin signaling pathway.

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