Investigation Of The Relationship Between Chromobox Homolog8and Nucleus Pulposus Cells Degeneration In Rat Intervertebral Disc

Aims: To investigate the expression of chromobox homolog8(CBX8) in nucleuspulposus (NP) cells from rat intervertebral disc (IVD) and its function in DNAdamage.Methods: NP cells were isolated from healthy rat intervertebral disc forimmunohistochemistry assay. CBX8siRNA was used for gene silencing, and reversetranscriptase-polymerase chain reaction (RT-PCR) was used to detect mRNAexpression of CBX8, type Ⅱ collagen, and proteoglycans. Cell proliferation and cellcycle were evaluated by MTT, colony-forming assay and flow cytometry. HydrogenPeroxide (H2O2) was used to simulate oxidative damage of DNA, and expression ofCBX8was examined using RT-PCR and Western blotting.Results: After5generations, mRNA expressions of type Ⅱ collagen andproteoglycans decreased（0.002±0.0006、0.24±0.02） with an increase of CBX8expression（1.65±0.33）. After CBX8was silenced using siRNA, the mRNAexpression of CBX8, type Ⅱ collagen and proteoglycans decreased（0.19±0.07、0.76±0.07、0.39±0.15）, CDKN2Awas increased(2.51±0.48）and the cell growth wasinhibited. Also, cell cycle was slowed down with most cells staying within G0/G1phases（84.27±1.25）%. Furthermore, CBX8expression increased facing to DNAoxidative damage simulated using H2O2. The data indicated that CBX8played an important role in cell proliferation and DNA damage.Conclusion: Cell proliferation and cycle were stimulated by CBX8, which may berelated to INK4A-ARF pathway. Moreover, CBX8functioned in DNA damage whichprovided it as a gene therapy target for disc degeneration treatment.