| Intervertebral disc degeneration (IVDD), which starts to develop since adolescence, can bring about a series of healthy problems, such as back pain, leg pain and short term or permanent dysfunction, giving rise to the high rate of degenerative disc disease and huge economic burdens. For the treatments of degenerative disc disease, conservative treatment and surgical treatment are mainly used and the problems cannot be solved fundamentally. Presently, the biological therapeutic strategies for IVDD are mainly focused on the transplantation of exogenous stem cells, which exhibits some unavoidable defects or complications, and therefore restricts the further study and clinical application of it.Recently, as the potential stem cell niches of the intervertebral disc (ISN) was detected in vivo, the theory of that the stem cells in the ISN (ISN-SCs) migrate into the inner part of the intervertebral disc may be one of the potential mechanisms for the disc regeneration was proposed, and facilitating the ISN-SCs migration-to-repair is becoming a new research direction of the biological therapies for IVDD. However, seldom study on the character identification of ISN-SCs and its potential mechanisms of migration-to-repair was reported according to our knowledge, which obstruct the development of the new biological strategies on the endogenous repair of the intervertebral disc.Hepatocyte growth factor (HGF), with the combination to its receptor’proto-oncogene MET coded transmembrane receptor tyrosine kinase’(c-met), can mediate various biological effects, such as promoting movement and proliferation of cells, facilitating growth and transfer of tumor cells, and regulating differentiation, adhesion, and migration of hematopoietic cells and stem cells.Based on the theoretical basis and research status mentioned above, this study aimed to isolate and culture the rat ISN-SCs, and to identify the characteristics of ISN-SCs with respect to the multipotential differentiation ability, the expression of stem cell related genes, and so on, which were compared with the bone marrow derived mesenchymal stem cells (BMSCs). Besides, the senescence model of nucleus pulposus cells (NPCs) was established and the repair effect of ISN-SCs on senescence NPCs were evaluated in protein and gene levels. Moreover, the relevant molecular mechanisms of HGF/c-met mediated ISN-SCs migration were further explored. These will contribute to the utilization of HGF on the repair of IVDD, provide more sufficient theoretical and research basis to further investigate the migration-to-repair effect of endogenous ISN-SCs on intervertebral disc (such as searching for the more appropriate migration chemokines), and inspire a new perspective on different biological approaches for the self-regeneration of intervertebral disc:promoting the migration of the endogenous stem cells.Part ⅠThe study on the isolation, culture and characteristics identification of ISN-SCsObjective:As the ISN and its related theories was proposed, the significant effect of ISN-SCs on the endogenous repair of intervertebral disc is gradually being taken seriously. However, further study on the ISN-SCs and its related endogenous repair was restricted because of the limitation knowledge of the ISN-SCs characteristics. The purposes of this study were to certify the existence of ISN-SCs in vitro and to identify the characteristics of ISN-SCs through comparing with those of BMSCs.Methods:Sprague-Dawley rats (male,10-week-old) were used as the experimental animals in this study. According to the anatomical areas, ISN tissues were detached by ophthalmic surgical instruments under the dissecting microscope. Primary ISN-SCs were harvested by digestion the ISN tissues in type Ⅱ collagen enzyme. BMSCs from the animals were harvested by density gradient centrifugation. Both of the cells were expanded in vitro and passage 4 (P4) cells were used for further analysis with respect to colony-forming ability, cell cycle, immunophenotype, stem cell-related gene expression, and proliferation and multipotential differentiation (osteogenesis, chondrogenesis, adipogenesis) abilities.Results:Both of ISN-SCs and MSCs met the minimal definition criteria of multipotent mesenchymal stromal cells (MSCs), with the characteristics of adhering to plastic, specific immunophenotype, and multipotent differentiation capacities. Particularly, ISN-SCs presented even greater potential in osteogenesis and chondrogenesis when compared to BMSCs. Besides, ISN-SCs also expressed comparable stem cell-related genes with those of BMSCs, and possessed colony-forming and self-renewal abilities.Conclusion:This in vitro study isolate and culture the ISN-SCs for the first time, which has determined the existence and characteristics of ISN-SCs. ISN-SCs belong to the MSC family and possess greater osteogenic and chondrogenic capacities than BMSCs. This finding is meaningful for further studies on investigating the migration of ISN-SCs into the intervertebral disc to facilitate endogenous repair, and may propose a new perspective on different biological approaches for intervertebral disc self-regeneration.Part IIThe study on the repair effect of ISN-SCs on senescence NPCsObjective:The purpose of this study was to confirm the repair effect of ISN-SCs on senescence NPCs.Methods:The ISN-SCs and NPCs from the experimental animals (Sprague-Dawley rats, male,10-week-old) were isolated and cultured. The senescence model of NPCs was established by continuous passage, and the effectiveness of the model was detected by the staining of senescence associated-β-galactosidase (SA-β-Gal). P4 ISN-SCs and senescence NPCs (1:1) were co-cultured in the cell-to-cell pattern for 7 days before being further tested, including toluidine blue staining for aggrecan, immumohistochemical staining for type II collagen, and quantitative real-time polymerase chain reaction (qPCR) for the mRNA expression level of ACAN and COL2al, which were compared with those of normal NPCs (P3) and senescence NPCs. Finally, the repair effect of ISN-SCs on senescence NPCs was determined.Results:SA-β-Gal staining indicated that NPCs gradually being senescence as the passage increased, with no obvious senescence cells in passage 3 (3.0±0.5%), less than 20% senescence cells in passage 5 (18.3±0.7%), and more than 80% senescence cells in passage 10 (86.0±4.6%). When compared to the normal NPCs (P3), the senescence NPCs (P10) expressed less aggrecan and type II collagen, and the mRNA expression level of ACAN and COL2al were significantly decreased (P< 0.05). At the end of the co-culture period, when compared to the senescence NPCs (P10), co-cultured cells expressed more aggrecan and type Ⅱ collagen (comparable to the normal NPCs), and the mRNA expression level of ACAN and COL2al were significantly increased (P< 0.05) and comparable to the normal NPCs (P> 0.05).Conclusion:The continuous passage method is capable of establishing the effective senescence model of NPCs, and P10 NPCs mainly meet the strict senescence criteria (number of senescence cells> 80%). ISN-SCs possess excellent repair effect on senescence NPCs, and can be utilized for further research on the repair of NPCs.Part ⅢThe study on the mechanisms of HGF/c-met mediated ISN-SCs migrationObjective:The purpose of this study was to further illuminate the cellular signaling pathway and associated molecular mechanisms of HGF/c-met mediated ISN-SCs migration.Method:The ISN-SCs and NPCs from the experimental animals (Sprague-Dawley rats, male, 10-week-old) were isolated and cultured. The senescence model of NPCs was established by continuous passage. Immunofluorescence (IF) and PCR were used to determine and compare the protein and mRNA expression level of HGF between normal and senescence NPCs. Also, the protein and mRNA expression level of c-met in ISN-SCs were examined. The enzyme linked immunosorbent assay (ELISA) was used to analyze and compare the exocrine level of HGF between normal and senescence NPCs. The influence of HGF on intracellular phosphorylation protein kinase (p-Akt) protein expression level was examined by western blot (WB), and HGF/c-met mediated PI3K/AKT signaling pathway was further confirmed by the intervention of c-met inhibitor (K-252a). The transwell migration system was used to clarify the influence of HGF/c-met mediated PI3K/AKT signaling pathway on the ISN-SCs migration by the intervention of c-met inhibitor (K-252a) and PI3K inhibitor (LY294002).Result:IF and PCR analysis showed that both of protein and mRNA of receptor c-met expressed extensively in the ISN-SCs; when compared to normal NPCs, senescence NPCs expressed significantly less protein and mRNA level of HGF (P< 0.05). The ELISA analysis indicated that the exocrine level of HGF in senescence NPCs was significantly decreased compared with that in normal NPCs (P< 0.05). The WB analysis revealed that HGF could significantly activated and increased the expression of p-Akt protein (P< 0.05), which was inhibited by the intervention of K-252a (with the expression of p-Akt protein significantly degreased, P< 0.05). The transwell migration analysis showed that HGF could promote ISN-SCs migration and significantly increased the number of migrated cells (P< 0.05), which could be inhibited significantly by K-252a and LY294002 (with the number of migrated cells decreased significantly, P< 0.05).Conclusion:The receptor c-met is extensively expressed in ISN-SCs, and the ligand HGF is extensively expressed in NPCs; Along with NPCs is becoming senescence, the expression and exocrine level of HGF decreases. While HGF/c-met promotes ISN-SCs migration through mediating the activation of PI3K/AKT signaling pathway. Therefore, the effect of promoting ISN-SCs migration in NPCs may decreases as the development of IVDD (with more proportion of senescence NPCs), which may be one of the important reasons contributing to the deteriorating process of IVDD. The results in this study laid a new theoretical basis for the application of HGF in the intervertebral disc regeneration:promoting ISN-SCs to migrate into the inner part of the disc. Also, sufficient research basis are proposed for further seeking for more appropriate and effective chemotactic factors, which will contribute to further study on the migration-to-repair effect of ISN-SCs in intervertebral disc. |
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