Overexpression Of N-Myc Downstream-regulated Gene 2 Regulates The Proliferation And Senescence Of Human Nucleus Pulposus Cell In Vitro

【Background】 Intervertebral disc degeneration(IDD) is the primary cause of spinal mechanical dysfunction and lumbocrural pain. The methods of IDD treatment include conservative treatment and excision plus fusion of intervertebral discs to improve clinical symptoms. However, none of these methods could reverse the progress of IDD. Although biological therapy of IDD is still in its early phase, it develops rapidly because of its potential long-term clinical benefit. The degeneration of nucleus pulposus cells(NPCs) cause the loss of proteoglycan and moisture, proliferation of fibrous tissue, spallation of anulus fibrosis, narrowing of intervertebral space and biomechanical dysfunction of the intervertebral disc. The newly discovered gene, human NDRG2, could be down-regulated in many tumor tissues. Recent researches find that NDRG2 is involved in the occurrence and development of tumorigenesis as well as the proliferation and differentiation of several cell types. In the current experiment, we speculate that NDRG2 may be involved in the regulation of NPCs proliferation and furthermore, the progress of IDD by regulating the senescence of NPCs 【Objectives】 This study was aimed to investigate the effect of N-myc downstream-regulated gene 2(NDRG2) on human nucleus pulposus(NP) cells in the area of cell senescence, and explore the role of NDRG2 in intervertebral disc degeneration(IDD). 【Methods】 1. The culture of human nucleus pulposus(NP) cells. 2. Human NP cells line were infected with Lenti-NDRG2 and Lenti-LacZ for use. 3. Real-time PCR was used to measure the positive expression of NDRG2 mRNA. 4. Western blot was used to measure the positive expression of NDRG2 protein. 5. Western blot was used to measure the positive expression of P53 and NDRG2 protein. 6. The proliferation of infected NP cells were determined with MTT. 7. Cell cycles were analyzed with flow cytometry. 8. Senescence associated-β-galactosidase staining was used to test the senescence of NP cells. 【Results】 1. Real-time PCR assays showed that the mRNA of NDRG2 was highly expressed in NP cells infected with Lenti-NDRG2 compared with the Lenti-Lacz group and blank group. 2. Western blot assays showed that NDRG2 was highly expressed in NP cells infected with Lenti-NDRG2 compared with the Lenti-Lacz group and blank group. 3. Western blot assays showed that NDRG2 was highly expressed in NP cells infected with Ad-P53 compared with the Ad-Lacz group and blank group. 4. The cell growth curve showed that the proliferation of NP cells was inhibited after infected with Lenti-NDRG2. 5. The flow cytometry result indicated that the S phase of Lenti-NDRG2 was significantly shorter than Lenti-Lacz group and blank group. 6. The positive percentage of SA-β-gal in the group of Lenti-NDRG2 was significantly higher than Lenti-Lacz group and blank group. 【Conclusion】Overexpression of NDRG2 could promote senescence of NP cells through regulating cell cycles and suppressing cell proliferation. NDRG2 was highly expressed in NP cells along with the expressed of P53. NDRG2 may be a target gene of P53 and may be involved in the P53 signal pathway in NP cells. NDRG2 may play a vital role in degeneration of intervertebral disc and this provides novel biological therapy for the intervertebral disc degeneration.