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Inhibition of the P53/P21 Pathway Attenuates the Effects of Senescent Nucleus Pulposus Cell‐Derived Exosomes on the Senescence of Nucleus Pulposus Cells

OBJECTIVE: The purpose of this paper is to investigate the effects of senescent nucleus pulposus cell (NPC)‐derived exosomes (SNPC‐Exo) and the roles of the P53/P21 pathway on the senescence of NPC. METHODS: The senescent phenotypes of NPC were induced by interleukin‐1β treatment. SNPC‐Exo was extra...

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Autores principales: Chen, Chang‐chun, Chen, Jing, Wang, Wen‐liang, Xie, Liang, Shao, Chuan‐qiang, Zhang, Yan‐xiang
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley & Sons Australia, Ltd 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7957423/
https://www.ncbi.nlm.nih.gov/pubmed/33314719
http://dx.doi.org/10.1111/os.12886
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author Chen, Chang‐chun
Chen, Jing
Wang, Wen‐liang
Xie, Liang
Shao, Chuan‐qiang
Zhang, Yan‐xiang
author_facet Chen, Chang‐chun
Chen, Jing
Wang, Wen‐liang
Xie, Liang
Shao, Chuan‐qiang
Zhang, Yan‐xiang
author_sort Chen, Chang‐chun
collection PubMed
description OBJECTIVE: The purpose of this paper is to investigate the effects of senescent nucleus pulposus cell (NPC)‐derived exosomes (SNPC‐Exo) and the roles of the P53/P21 pathway on the senescence of NPC. METHODS: The senescent phenotypes of NPC were induced by interleukin‐1β treatment. SNPC‐Exo was extracted from the culture medium of senescent NPC and purified by differential centrifugation. The structure of SNPC‐Exo was identified by transmission electron microscopy and western blot analysis was used to determine the exosomal marker proteins CD63 and Tsg101. Western blot analysis was performed to determine the relative expression levels of P16, P21, and P53 in NPC. Senescence‐associated β‐galactosidase (SA‐β‐gal) staining was used to stain the senescent NPC and a phase contrast microscope was used to observe and count the SA‐β‐gal staining of NPC. The proliferation of SNPC‐Exo‐treated NPC was assessed using growth curve analysis and the colony formation assay. The cell cycle of SNPC‐Exo‐treated NPC was determined by flow cytometry. NPC were transfected with siRNA to knock down P53 and P21 expression. RESULTS: Interleukin‐1β‐treated NPC had a higher percentage of SA‐β‐gal positive cells (45%) than the control group (20%) and showed an increase in the relative expression of P16, P21, and P53 (P < 0.05). SNPC‐Exo were positive for exosomal marker protein CD63 and Tsg 101 and negative for calnexin, and successfully internalized as previously described. SNPC‐Exo‐treated NPC showed an increase in the relative expression of P21 and P53 (P < 0.05). Compared with the control group, the SNPC‐Exo‐treated NPC showed a lower growth rate (3 times lower on the 5th day and 2 times lower on the 7th day), fewer colony‐forming units (12.0%), and a higher percentage of SA‐β‐gal‐positive NPC (50.0%). The SNPC‐Exo‐treated NPC contained more G1 phase cells (68.0%) and fewer S phase (15.5%) cells than the control group (53.0% in G1 phase, 33.5% in S phase). The expression of P21 and P53 significantly decreased in SNPC‐exo‐treated NPC after siRNA transfection (P < 0.05), followed by a higher growth rate (2 times higher on the 5th day and 1.5 times higher on the 7th day) and lower percentage of SA‐β‐gal‐positive NPC (22.5%). Moreover, the inhibition of the P53/P21 pathway promoted the SNPC‐Exo‐treated NPC to enter the S phase (from 15.5% to 25.3%). CONCLUSION: The inhibition of the P53/P21 pathway attenuated the senescence of NPC induced by SNPC‐Exo.
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spelling pubmed-79574232021-03-19 Inhibition of the P53/P21 Pathway Attenuates the Effects of Senescent Nucleus Pulposus Cell‐Derived Exosomes on the Senescence of Nucleus Pulposus Cells Chen, Chang‐chun Chen, Jing Wang, Wen‐liang Xie, Liang Shao, Chuan‐qiang Zhang, Yan‐xiang Orthop Surg Scientific Articles OBJECTIVE: The purpose of this paper is to investigate the effects of senescent nucleus pulposus cell (NPC)‐derived exosomes (SNPC‐Exo) and the roles of the P53/P21 pathway on the senescence of NPC. METHODS: The senescent phenotypes of NPC were induced by interleukin‐1β treatment. SNPC‐Exo was extracted from the culture medium of senescent NPC and purified by differential centrifugation. The structure of SNPC‐Exo was identified by transmission electron microscopy and western blot analysis was used to determine the exosomal marker proteins CD63 and Tsg101. Western blot analysis was performed to determine the relative expression levels of P16, P21, and P53 in NPC. Senescence‐associated β‐galactosidase (SA‐β‐gal) staining was used to stain the senescent NPC and a phase contrast microscope was used to observe and count the SA‐β‐gal staining of NPC. The proliferation of SNPC‐Exo‐treated NPC was assessed using growth curve analysis and the colony formation assay. The cell cycle of SNPC‐Exo‐treated NPC was determined by flow cytometry. NPC were transfected with siRNA to knock down P53 and P21 expression. RESULTS: Interleukin‐1β‐treated NPC had a higher percentage of SA‐β‐gal positive cells (45%) than the control group (20%) and showed an increase in the relative expression of P16, P21, and P53 (P < 0.05). SNPC‐Exo were positive for exosomal marker protein CD63 and Tsg 101 and negative for calnexin, and successfully internalized as previously described. SNPC‐Exo‐treated NPC showed an increase in the relative expression of P21 and P53 (P < 0.05). Compared with the control group, the SNPC‐Exo‐treated NPC showed a lower growth rate (3 times lower on the 5th day and 2 times lower on the 7th day), fewer colony‐forming units (12.0%), and a higher percentage of SA‐β‐gal‐positive NPC (50.0%). The SNPC‐Exo‐treated NPC contained more G1 phase cells (68.0%) and fewer S phase (15.5%) cells than the control group (53.0% in G1 phase, 33.5% in S phase). The expression of P21 and P53 significantly decreased in SNPC‐exo‐treated NPC after siRNA transfection (P < 0.05), followed by a higher growth rate (2 times higher on the 5th day and 1.5 times higher on the 7th day) and lower percentage of SA‐β‐gal‐positive NPC (22.5%). Moreover, the inhibition of the P53/P21 pathway promoted the SNPC‐Exo‐treated NPC to enter the S phase (from 15.5% to 25.3%). CONCLUSION: The inhibition of the P53/P21 pathway attenuated the senescence of NPC induced by SNPC‐Exo. John Wiley & Sons Australia, Ltd 2020-12-13 /pmc/articles/PMC7957423/ /pubmed/33314719 http://dx.doi.org/10.1111/os.12886 Text en © 2020 The Authors. Orthopaedic Surgery published by Chinese Orthopaedic Association and John Wiley & Sons Australia, Ltd. This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.
spellingShingle Scientific Articles
Chen, Chang‐chun
Chen, Jing
Wang, Wen‐liang
Xie, Liang
Shao, Chuan‐qiang
Zhang, Yan‐xiang
Inhibition of the P53/P21 Pathway Attenuates the Effects of Senescent Nucleus Pulposus Cell‐Derived Exosomes on the Senescence of Nucleus Pulposus Cells
title Inhibition of the P53/P21 Pathway Attenuates the Effects of Senescent Nucleus Pulposus Cell‐Derived Exosomes on the Senescence of Nucleus Pulposus Cells
title_full Inhibition of the P53/P21 Pathway Attenuates the Effects of Senescent Nucleus Pulposus Cell‐Derived Exosomes on the Senescence of Nucleus Pulposus Cells
title_fullStr Inhibition of the P53/P21 Pathway Attenuates the Effects of Senescent Nucleus Pulposus Cell‐Derived Exosomes on the Senescence of Nucleus Pulposus Cells
title_full_unstemmed Inhibition of the P53/P21 Pathway Attenuates the Effects of Senescent Nucleus Pulposus Cell‐Derived Exosomes on the Senescence of Nucleus Pulposus Cells
title_short Inhibition of the P53/P21 Pathway Attenuates the Effects of Senescent Nucleus Pulposus Cell‐Derived Exosomes on the Senescence of Nucleus Pulposus Cells
title_sort inhibition of the p53/p21 pathway attenuates the effects of senescent nucleus pulposus cell‐derived exosomes on the senescence of nucleus pulposus cells
topic Scientific Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7957423/
https://www.ncbi.nlm.nih.gov/pubmed/33314719
http://dx.doi.org/10.1111/os.12886
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