抑制 P53/P21 通路可减轻衰老的髓核细胞来源的外泌体对髓核细胞衰老的影响。
Inhibition of the P53/P21 Pathway Attenuates the Effects of Senescent Nucleus Pulposus Cell-Derived Exosomes on the Senescence of Nucleus Pulposus Cells.
机构信息
Department of Orthopaedics, Baoji Central Hospital, Baoji, China.
Medical School, Baoji Vocational Technology College, Baoji, China.
出版信息
Orthop Surg. 2021 Apr;13(2):583-591. doi: 10.1111/os.12886. Epub 2020 Dec 13.
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.
目的
本文旨在研究衰老核髓核细胞（NPC）来源的外泌体（SNPC-Exo）的作用，以及 P53/P21 通路在 NPC 衰老中的作用。
方法
用白细胞介素-1β处理诱导 NPC 衰老表型。通过差速离心从衰老 NPC 的培养基中提取 SNPC-Exo，并通过透射电子显微镜鉴定 SNPC-Exo 的结构，用 Western blot 分析确定外泌体标记蛋白 CD63 和 Tsg101。Western blot 分析 NPC 中 P16、P21 和 P53 的相对表达水平。用衰老相关β-半乳糖苷酶（SA-β-gal）染色法染色衰老的 NPC，用相差显微镜观察和计数 NPC 的 SA-β-gal 染色。用生长曲线分析和集落形成实验评估 SNPC-Exo 处理的 NPC 的增殖情况。用流式细胞术测定 SNPC-Exo 处理的 NPC 的细胞周期。用 siRNA 转染 NPC 以敲低 P53 和 P21 的表达。
结果
白细胞介素-1β处理的 NPC 中 SA-β-gal 阳性细胞的比例（45%）明显高于对照组（20%），且 P16、P21 和 P53 的相对表达水平升高（P<0.05）。SNPC-Exo 阳性表达外泌体标记蛋白 CD63 和 Tsg101，而阴性表达 calnexin，且能够像之前报道的那样内化。SNPC-Exo 处理的 NPC 中 P21 和 P53 的相对表达水平升高（P<0.05）。与对照组相比，SNPC-Exo 处理的 NPC 生长速度较低（第 5 天降低 3 倍，第 7 天降低 2 倍），集落形成单位较少（12.0%），SA-β-gal 阳性 NPC 的比例较高（50.0%）。SNPC-Exo 处理的 NPC 中 G1 期细胞较多（68.0%），S 期细胞较少（G1 期 53.0%，S 期 33.5%）。SNPC-Exo 处理的 NPC 经 siRNA 转染后 P21 和 P53 的表达明显降低（P<0.05），随后生长速度加快（第 5 天增加 2 倍，第 7 天增加 1.5 倍），SA-β-gal 阳性 NPC 的比例降低（22.5%）。此外，抑制 P53/P21 通路促进了 SNPC-Exo 处理的 NPC 进入 S 期（从 15.5%增加到 25.3%）。
结论
抑制 P53/P21 通路可减轻 SNPC-Exo 诱导的 NPC 衰老。
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