Objective To summarize the role of cellular senescence and senescent secretary phenotype in the intervertebral disc (IVD) degeneration. Methods Relevant articles that discussed the roles of cellular senescence in the IVD degeneration were extensively reviewed, and retrospective and comprehensive analysis was performed. The senescent phenomenon during IVD degeneration, senescent secretary phenotype of the disc cells, senescent pathways within the IVD microenvironment, as well as the anti-senescent approaches for IVD regeneration were systematically reviewed. Results During aging and degeneration, IVD cells gradually and/or prematurely undergo senescence by activating p53-p21-retinoblastoma (RB) or p16INK4A-RB senescent pathways. The accumulation of senescent cells not only decreases the self-renewal ability of IVD, but also deteriorates the disc microenvironment by producing more inflammatory cytokines and matrix degrading enzymes. More specific senescent biomarkers are required to fully understand the phenotype change of senescent disc cells during IVD degeneration. Molecular analysis of the senescent disc cells and their intracellular signaling pathways are needed to get a safer and more efficient anti-senescence strategy for IVD regeneration. Conclusion Cellular senescence is an important mechanism by which IVD cells decrease viability and degenerate biological behaviors, which provide a new thinking to understand the pathogenesis of IVD degeneration.
Objective To observe the replicative senescence of rat articular chondrocyte cultured in vitro so as to provide reference for the succeeding experiment of using medicine interfere and reverse the cataplasia of tissue engineering cartilage or probing cataplasia mechanism．Methods Different generations(P1, P2, P3 and P4) of the chondrocytes were detected with the methods of histochemistry for β-galactosidase (β-gal)， electronmicroscope for ultromicrostructure， immunocytochemistry for proliferating cell nuclear antigen (PCNA)，alcian blue stain for content and structure of sulfatglycosaminoglycan (GAG) of extracellular matrix (ECM)，reverse transcriptionpolymerase chain reaction (RTPCR) for content of collagen Ⅱ，flow cytometry for cell life cycle and proliferative index(PI) to observe senescence of chondrocytes．Results In the 4th passage，the chondrocytes emerging quantitively positive express of β-gal，cyto-architecture cataplasia such as caryoplasm ratio increasing and karyopycnosis emerging under electronmicroscope ，cell life cycle being detented on G1 phase(83．8％)，while in P1, P2, P3 the content of G1 phase was 79.1%, 79.2%, 80.8% respectively. In the 4th passage, PI decreased(16．2％)，while in P1, P2, P3, it was 20.9%, 20.8%, 19.2%. The positive percentage of PCNA，the content of GAG(long chain molecule) and the positive expression of collagen Ⅱ diminished,all detections above were significantly different (Plt;0.01) when compared the 4th passage with the preceding passages．Conclusion Chondrocytes show the onset of senescence in the 4th passage．
Objective To study morphological and biological senescence changes induced by D-galactose in the cultured rat mesenchymal stem cells. Methods After 3rd generations cultured in the DMEM-F12, MSCs were changed into DMEMF12 medium containing 8 g/L D-galatose and cultured to the 6th generations as the inducement group. The comparison were the 6thgenerations which was cultured in the DMEM-F12 medium all along, and then indentified by surface wave. Using flow cytometer to check the comparisons cell cycle change after swing in with 8 g/L D-galatose within the 4 days. In the first 7daysto draw the growth curve to the two groups. Optical and electronic microscope were used to identify the influences of characteristic morphological of mesenchymal stem cells of the two groups, the influences of biological markers were identified by single cell gel electrophoresis and β-galactose dye. Results After treatment with D-galactose, the mesenchymal stem cells displayed morphological and biological changes in the cell senescence with the senescent characteristic morphological markers; 85% of the cells were X-gal dye masculine, and the singal cell gel electrophoresis showed DNA damnification. The flow cytometry showed that 90% of the cells stayed in G 0/G 1, but the cells in S and G 2/M almost disappeared.However, the cells in the control group had no such DNA damages. Conclusion D-galactose can induce senescence of the mesenchymal stem cells, and 8 g/L is the best concentration to do so. This study has provided a good model forthe research of the mesenchymal stem cells senescence.
OBJECTIVE: To explore the SV40-mediated immortalization, the related factors and their roles in cell immortalization. METHODS: The original articles about cell immortalization and replicative senescence in recent decade were reviewed. RESULTS: Cell immortalization was a multifaceted phenomenon, it was involved in viral DNA integration, activation of telomerase, inactivation of growth suppressors, and so on, and their roles were closely related. CONCLUSION: The research on cell immortalization may be expected to provide important insights into a broad range of cellular biological phenomenon, and the immortalized cells can play important roles in the research of cell engineering and tissue engineering as standard cells.
ObjectiveTo investigate the expression of p16INK4a in nucleus pulposus (NP) and to clarify its relationship with intervertebral disc degeneration so as to provide evidence for biological repair of intervertebral disc. MethodsThe NP specimens were obtained from 17 patients with intervertebral disc degeneration undergoing discectomy, who aged 40-50 years (mean, 45.4 years). Based on the preoperative MRI, there were 10 cases of grade Ⅲ degeneration, and 7 cases of grade IV degeneration. Cell senescence was evaluated by detecting senescence-associated β-galactosidase (SA-β-gal) activity. Senescence marker (p16INK4a) and disc degeneration markers [A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS 5), Aggrecan, and Sryrelated HMG box transcri ption factor 9 (Sox-9)] were determined in the NP specimens with immunohistochemistry and Western blot. The correlation between ADAMTS 5 and p16INK4a was analyzed. ResultsClustered distribution of green SA-β-gal-positive cells was seen in the NP with grade Ⅲ and IV degeneration. A few single round SA-β-gal-positive NP cells (NPCs) wrapped by the layered extracellular matrix were also seen in the NP with grade Ⅲ degeneration. It was difficult to see single distribution of NPCs in the NP with grade IV degeneration. The percentage of SA-β-gal-positive cells was 22.7%±5.4% and 37.1%±7.6% in the NP with grade Ⅲ and IV degeneration respectively, showing significant difference (t=-9.666, P=0.000). The percentages of p16INK4a-positive and ADAMTS 5-positive NPCs in the NP with grade IV degeneration were significantly higher than those with grade Ⅲ degeneration (P<0.05). The percentages of Aggrecan-positive and Sox-9-positive NPCs in the NP with grade IV degeneration were significantly lower than those in the NP with grade Ⅲ degeneration (P<0.05). The protein expressions of Aggrecan and Sox-9 in the NP with grade IV degeneration were significantly lower than those in the NP with grade Ⅲ degeneration (P<0.05). The NP with grade IV degeneration showed significantly higher protein expressions of p16INK4a and ADAMTS 5 (P<0.05). Importantly, there was a good correlation between p16INK4a and ADAMTS 5 protein expressions (r=0.908, P=0.000). ConclusionPremature senescent NPCs increase in the NP with the advancing disc degeneration. The expression of p16INK4a and its association with degeneration grades suggest that the p16INK4a may play a significant role in the pathogenesis of intervertebral disc degeneration.
ObjectiveTo investigate the biological characteristics of bone marrow mesenchymal stem cells (BMSCs) in microenvironment of premature senescence of nucleus pulposus cells (NPCs) so as to lay a foundation for the repair of intervertebral disc degeneration by BMSCs transplantation. MethodsHuman degenerative nucleus pulposus and normal bone marrow were collected, and then NPCs and BMSCs were isolated, cultured, and identified. The 3rd passage BMSCs and the 1st passage NPCs with premature senescence were co-cultured without contact in the Transwell culture system. NPCs to BMSCs ratio was 75%:25% (group A), 50%:50% (group B), and 0:100% (group C). The morphological changes of BMSCs were observed by inverted phase contrast microscopy and transmission electron microscopy. At 3 and 6 days after co-culture, cell counting kit 8 was used to detect cell viability, flow cytometry was used to observe the cell cycle and detect DNA metabolism after BrdU labeling. Cell senescence was also evaluated by detecting senescence associated β-galactosidase (SA-β-gal) activity. ResultsThe typical morphology of cell senescence was seen in groups A and B, especially in group A. At 3 and 6 days after co-culture, the cell survival rate of group A was significantly lower than that of group B (P<0.05). At 3 days after co-culture, the proportion of cells in G1 phase in group A was significantly higher than that in groups B and C (P<0.05), the proportion of cells in S phase in group A was significantly lower than that in groups B and C (P<0.05). At 6 days, the proportion of cells in G1 phase in group A was about 81.0%, and the proportion of cells in S phase and G2 phase decreased, showing significant difference when compared with groups B and C (P<0.05); the proportion of cells in G1 phase in group B was about 74.4%, showing significant difference when compared with group C (P<0.05). BrdU content in group A was significantly lower than that in groups B and C at 3 and 6 days after co-culture (P<0.05), but no significant difference was found between groups B and C at 3 days (P>0.05); Brdu content in group B was also significantly reduced when compared with group C (P<0.05) at 6 days. At 6 days, SA-β-gal activity was significantly increased in groups A and B, and significant difference was shown in SA-β-gal positive cell number between groups (P <0.05). ConclusionPremature senescence of NPCs can down-regulate the proliferation capacity of co-cultured BMSCs by the paracrine effect. The greater proportion of NPCs with premature senescence is, the earlier senescence of BMSCs will be induced.
ObjectiveTo review the pathological effects of cellular senescence in the occurrence and development of osteoarthritis (OA) and potential therapeutic targets.MethodsThe role of chondrocyte senescence, synovial cell senescence, mesenchymal stem cells senescence in OA, and the biological mechanism and progress of chondrocyte senescence were summarized by consulting relevant domestic and abroad literature.ResultsThe existing evidence has basically made clear that chondrocyte senescence, mesenchymal stem cells senescence, and cartilage repair abnormalities, and the occurrence and development of OA have a certain causal relationship, and the role of the senescence of synovial cells, especially synovial macrophages in OA is still unclear. Transcription factors and epigenetics are the main mechanisms that regulate the upstream pathways of cellular senescence. Signal communication between cells can promote the appearance of senescent phenotypes in healthy cells. Targeted elimination of senescent cells and promotion of mesenchymal stem cells rejuvenation can effectively delay the progress of OA.ConclusionCellular senescence is an important biological phenomenon and potential therapeutic target in the occurrence and development of OA. In-depth study of its biological mechanism is helpful to the early prevention and treatment of OA.
Cell senescence is a state of irreversible cell cycle arrest and simultaneously secretes inflammatory factors, chemokines and other senescence-associated secretory phenotype (SASP), which plays an important role in the progression of kidney diseases, metabolic diseases and other diseases. Renal tubular cell (RTC) senescence is a key cellular biological event in the progression of acute kidney injury (AKI). Senescent RTCs not only inhibit the regeneration and repair of AKI, but also release SASP to promote the progression of AKI. Inhibition of RTC senescence, targeted removal of senescent RTCs or promotion of senescent RTCs apoptosis could improve the prognosis of AKI, indicating that these methods have broad application prospects.
Objective To investigate the effect of epigallocatechin gallate (EGCG) on chondrocyte senescence and its mechanism. Methods The chondrocytes were isolated from the articular cartilage of 4-week-old Sprague Dawley rats, and cultured with type Ⅱcollagenase and passaged. The cells were identified by toluidine blue staining, alcian blue staining, and immunocytochemical staining for type Ⅱ collagen. The second passage (P2) cells were divided into blank control group, 10 ng/mL IL-1β group, and 6.25, 12.5, 25.0, 50.0, 100.0, and 200.0 μmol/L EGCG+10 ng/mL IL-1β group. The chondrocyte activity was measured with cell counting kit 8 after 24 hours of corresponding culture, and the optimal drug concentration of EGCG was selected for the subsequent experiment. The P2 chondrocytes were further divided into blank control group (group A), 10 ng/mL IL-1β group (group B), EGCG+10 ng/mL IL-1β group (group C), and EGCG+10 ng/mL IL-1β+5 mmol/L 3-methyladenine (3-MA) group (group D). After cultured, the degree of cell senescence was detected by β-galactosidase staining, the autophagy by monodansylcadaverine method, and the expression levels of chondrocyte-related genes [type Ⅱ collagen, matrix metalloproteinase 3 (MMP-3), MMP-13] by real-time fluorescent quantitative PCR, the expression levels of chondrocyte-related proteins (Beclin-1, LC3, MMP-3, MMP-13, type Ⅱ collagen, P16, mTOR, AKT) by Western blot. Results The cultured cells were identified as chondrocytes. Compared with the blank control group, the cell activity of 10 ng/mL IL-1β group significantly decreased (P<0.05). Compared with the 10 ng/mL IL-1β group, the cell activity of EGCG+10 ng/mL IL-1β groups increased, and the 50.0, 100.0, and 200.0 μmol/L EGCG significantly promoted the activity of chondrocytes (P<0.05). The 100.0 μmol/L EGCG was selected for subsequent experiments. Compared with group A, the cells in group B showed senescence changes. Compared with group B, the senescence rate of chondrocytes in group C decreased, autophagy increased, the relative expression of type Ⅱ collagen mRNA increased, and relative expressions of MMP-3 and MMP-13 mRNAs decreased; the relative expressions of Beclin-1, LC3, and type Ⅱ collagen proteins increased, but the relative expressions of P16, MMP-3, MMP-13, mTOR, and AKT proteins decreased; the above differences were significant (P<0.05). Compared with group C, when 3-MA was added in group D, the senescence rate of chondrocytes increased, autophagy decreased, and the relative expressions of the target proteins and mRNAs showed an opposite trend (P<0.05). ConclusionEGCG regulates the autophagy of chondrocytes through the PI3K/AKT/mTOR signaling pathway and exerts anti-senescence effects.
Objective To explore the role of hydrogen peroxide (H2O2) in inducing chronic oxidative stress in microglia aging. Methods BV2 microglia purchased from ATCC in less than 10 generations were treated with 0, 50, 100, 200 μmol/L H2O2 at different concentrations. According to the concentration of H2O2 used, the BV2 microglia were divided into a control group and H2O2 -50 μmol/L Group, H2O2 -100 μmol/L Group, H2O2 -200 μmol/L Group. Cell proliferation was measured by CCK8 cell proliferation assay. Age-related β-galactosidase (SA-β-gal) staining assay, and expression of age-related cyclin molecules p16, p21, p53 and senescence sssociated secretory phenotype interleukin 1 beta (IL-1β), transforming growth factor-β (TGF-β) and matrix metalloprotein 9 (MMP9) detected by quantitative real-time polymerase chain reaction were used to measure celluar senescence. Results During the induction process, H2O2-200 μmol/L caused significant damage to BV2 microglia, therefore no subsequent testing was conducted. Finally, the control group, H2O2-50 μmol/L group and H2O2-100 μmol/L group cells were collected. The differences in cell survival rate (F=46.176, P<0.001) and positive rate of SA-β-gal staining (F=553.1, P<0.001) among the three groups were statistically significant. The cell survival rate of H2O2-50 μmol/L group had no significant change (P>0.05), while the cell survival rate of H2O2-100 μmol/L group decreased significantly (P<0.001). The positive rate of SA-β-gal staining in H2O2-50 μmol/L group and H2O2-100 μmol/L group was increased (P<0.001), and the positive rate of SA-β-gal staining in H2O2-100 μmol/L group was higher than that in H2O2-50 μmol/L group (P<0.001). The mRNA levels of senescence related cyclin molecules p16, p21 and p53 were up-regulated under the induction of 50, 100 μmol/L H2O2 (P<0.05), and the expressions of IL-1β, TGF-β and MMP9 of senescence associated secretory phenotype were increased (P<0.05). The increase of H2O2-50 μmol/L group was more obvious (P<0.05). Conclusion The aging model of BV2 microglia can be successfully established by inducing 8 d with 100 μmol/L H2O2, and the mechanism may be related to promoting the secretion of p16, p21, p53, IL-1β, TGF-β and MMP9.