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Objective: To observe human bone marrow mesenchymal stem cells (human bone marrow stromal cells, hBMSCs) copy the aging process as well as the aging process hBMSCs biological characteristics, and to explore the role of the 20S proteasome in the in the hBMSCs replicative senescence process, which reveals the the hBMSCs replicative senescence mechanism to provide guidance on the basis of the hBMSCs the clinical application. Methods: Cultured hBMSCs, based on the number of passage cells were divided into early (1-3 generations), medium-term (7-9 generation) and late (12-14 generations): (1) inverted phase contrast microscope in different periods The morphological characteristics of the beta-galactosidase enzyme staining of cell senescence process, thiazolyl tetrazolium (MTT) colorimetric assay and bromine deoxy-uridine (bromodeoxyuridine BrdU) incorporation assay cell proliferation. (2) immunofluorescence staining 20S proteasome in hBMSCs replicative aging process in expression. (3) 10μM specific proteasome inhibitor MG132 role in the early hBMSCs daily role 2h, repeated stimulation 4d, followed by β-galactosidase staining was observed changes in senescence marker beta-galactosidase expression, and apply the MTT assay and BrdU incorporation assay cell proliferation activity detection proteasome inhibitor early hBMSCs proliferation of. Results: (1) early hBMSCs cell body smaller, clear nucleolus, spindle-shaped cells, like fibroblast-like shape. Accompanied by an increase in vitro passage number of hBMSCs cell body, protruding increased, reduced cell diopter, many small particles in the cytoplasm, the ability to increase cell adhesion, growth slowed. Signs of aging substance beta-galactosidase enzyme staining showed positive cells in the early, middle and late group rate of 9% ± 7%, 54% ± 8%, 90% ± 8%, among the three groups comparison p LT; 0.05, suggesting that hBMSCs presence of replicative senescence in vitro. MTT assay to detect different stages of culture the hBMSCs cell viability early group OD value of 0.61 ± 0.04, interim group to 0.53 ± 0.05 late group was 0.37 ± 0.03, among the three groups comparison p LT; 0.05, indicating that replicative senescence process in cell vitality significantly reduced, at the same time BrdU incorporation experimental results also show that the late group cells BrdU-positive rate (8% ± 2%) compared with the early group (73% ± 11%) was significantly reduced, and p lt; 0.05, further showed that in the copy hBMSCs proliferation activity was reduced in the process of senescence. (2) immunofluorescence staining showed that hBMSCs expression of 20S proteasome, in early and mid group 20S proteasome positive rate of 100%, however, advanced cell 20S proteasome expression level of the positive rate to 31%, suggesting that proteasome activity The hBMSCs replicative senescence may reduce. (3) Early hBMSCs by MG132 role after beta-galactosidase staining enhanced beta-galacto-glucosidase positive rate of 87% ± 13.2%, compared with DMSO (control group) (19% ± 6.8%) was significantly higher , and p lt; 0.05, MTT detection results confirmed that the the MG132 group of OD value (0.36 ± 0.06) compared with DMSO group (0.47 ± 0.08) was significantly reduced, and the p lt; 0.05 and BrdU incorporation experiments also display MG132 group Brdu positive rate ( 7% ± 2%) than the control group (54% ± 4%) was significantly decreased, and p lt; 0.05, show that proteasome inhibitors can induce early hBMSCs aging phenotype, further proteasome activity decreased with hBMSCs copy senescence. Conclusion: (1) hBMSCs in vitro replicative senescence; (2) 20S proteasome expression level of hBMSCs replicative senescence process. (3) application of a specific inhibitor of the proteasome can induce the hBMSCs showing senescent phenotype, suggesting that proteasome activity decreased hBMSCs aging.
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