| Objective To investigate the effects of hypoxic preconditioning of human adipose-derived stem cells(ADSCs)and normoxic preconditioning of human adipose-derived stem cells on the aging of human dermal fibroblasts(HDFs).Methods Normal adult subcutaneous adipose tissue and dermal tissue were taken.Adipose tissue was digested by enzymes,dermal tissue was separated and cultured in vitro by tissue block method.Human adipose-derived stem cells(ADSCs)and dermal fibroblasts(HDFs)were identified.The cells were cultured and frozen to meet the needs of the experiment.Human adipose-derived stem cells were subcultured in hypoxic workstation(1% O 2,5% CO 2,94% N 2,37 C)and normal incubator(21% O 2,5% CO 2,the rest of nitrogen balance,37 C).After 48 hours of treatment,they were cultured in groups for experimental study.The experiment was designed into three groups: blank control group,experimental group I,and experimental group II.In the blank control group,dermal fibroblasts were cultured alone;in the experimental group I,the normal oxygen group and in the experimental group II,the hypoxia group.In both groups,a non-contact indirect co-culture model between ADSCs and HDFs was constructed by hanging cell culture dish(Transwell chamber).Fibroblasts were implanted in the lower chamber,adipose stem cells cultured in the upper chamber,and hypoxia in the experimental group II.Adipose stem cells were cultured in workstation.The three groups of cells were cultured in ordinary incubator for 3 days,and then the aging indexes of dermal fibroblasts were detected.Aging indicators were detected as follows: A.Aging-related beta-galactosidase(SA-beta-gal)staining was performed to detect the aging of fibroblasts;B.WST-1 method was used to determine the activity of superoxide dismutase(SOD)in fibroblasts;C.TBA method was used to determine the content of micromalondialdehyde(MDA)in fibroblasts;D.ELISA method was used to determine vascular endothelial growth factor(VEGF)concentration in fibroblasts;E.Real-time PCR was used to detect the expression of aging-related proteins p16,p21,p53 in fibroblasts.Result 1.? Adipose stem cells extracted from normal human adipose tissue grew in long spindle shape.Flow cytometry showed that the expression of surface antigens CD73 and CD90 of adipose stem cells was positive,while the expression of CD34 and CD45 was negative.Induced adipose-derived stem cells can successfully differentiate into adipogenic and osteogenic cells.? Dermal fibroblasts extracted from human dermal tissue grew in spindle,strip,polygon or irregular shape.The expression of specific protein(fsp-1)and vimentin was positive by immunofluorescence and keratin was negative.? After co-culture of ADSCs and HDFs in experimental group I and II,we can see that both of them have fibroblast-like morphology in naked view,and there is no obvious morphological difference.2.According to the positive rate of SA-beta-Gal staining,the activity of SOD,the content of MDA,the concentration of vascular endothelial growth factor and the expression level of senescence-related indicators by RT-PCR,the senescence indicators of blank control group were higher than those of experimental group I,the senescence indicators of experimental group I were higher than those of experimental group II.The aging index of blank control group was significantly higher than that of experimental group II,and the difference was statistically significant.Conclusion In this experiment,human adipose-derived stem cells and human dermal fibroblasts were co-cultured indirectly by hypoxic preconditioning in vitro to detect the aging degree of dermal fibroblasts.Molecular cell biology tests showed that: 1.Human adipose-derived stem cells pretreated with hypoxia had higher biological activity than human adipose-derived stem cells cultured in normoxia.2.ADSCs have a certain anti-aging effect on HDFs,and the anti-aging effect of ADSCs under hypoxic environment is more significant.Therefore,hypoxic preconditioning of ADSCs can reduce the biological indicators related to cell aging. |
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