Differentiation And Immortalization Of Bone Marrow Strom Cells Toward To Nucleus Pulposus Cell-Like Cells By Co-cultured With Nucleus Pulposus Cell

PartⅠ:The Pathology and Imaging Performance of Rabbit Disc Degeneration Model Induced by the Aspiration of Nucleus PulposusObjective:To investigate the use of liposuction to build rabbit model of disc degeneration and to observe the performance of its pathology and imaging.Methods:Healthy 10-month-old New Zealand rabbits 28,7 for control group and 21 for experimental group.21-gauge hypodermic needle was used to aspirate nucleus pulposus from L4/5 disc about 0.008～0.012g. All the experimental rabbits were continued to foster for 4-20 weeks. X-ray plain film, MRI and Alcian blue staining were used to observe the organizational changes in the intervertebral disc degeneration.Results:The percentage of disc height index (DHI%) of control group,4-week group, 12-week group and 20-week group respectively was (100±0)%, (81±3.2)%, (75±2.5)% and (71±1.8)% (P<0.05). T2-weighted image showed that the signal of the discs was significantly decreased, and the content of aggrecan was also significantly decreased.Conclusion:The puncture and aspiration is a reliable and convenient way to construct rabbit models of intervertebral disc degeneration, and these models can be used for the research of human the mechanism and clinical therapeutic research. PartⅡ:The Induction of Nucleus Pulposus Cells to Bone Marrow Stem Cells by Co-culturedObjective:To research the effect of nucleus pulposus cells to bone marrow stem cells which were encapsulated in alginate beads when they were co-cultured in vitro.Methods:①we got the primary nucleus pulposus cells and bone marrow stem cells from 5 healthy New Zealand rabbits of 4-month-old, and the nucleus pulposus cells and bone marrow stem cells were selected by their adherence to tissue culture flasks.②Cultures of MSCs were trypsinized, centrifuged, and mixed homogeneously into 1.2% alginate solution at 106 cells/mL. The alginate solution was injected into 3.5% CaCl2 solution by injector forming small droplets of cell-alginate beads.③The nucleus pulposus cells and cell-alginate beads were co-cultured in Six-well plates. At the 7th day and the 15th day, we collected the bone marrow stem cells from the cell-alginate beads. Immunohistochemical techniques, rt-PCR and Western blot technique was used to study the expression of collagen-Ⅱand aggrecan.Results:The immunohistochemical stain show that collagen-Ⅱand aggrecan has been expressed in the MSCs, and we also find that both of the collagen-Ⅱand aggrecan, our purpose strips of the 15th day's rt-PCR result are brighter than those of the 7th day's rt-PCR result.Conclusion:In vitro, bone marrow stem cells can be differentiated into the nucleus pulposus cell-like cells by been co-cultured with the nucleus pulposus cells. It can be served as optimal cell source for therapy of the degeneration disc. PartⅢ:Differentiation and Immortalization of Bone Marrow Strom Cells toward to Nucleus Pulposus Cell-Like CellsObjective:To attempt to use the way of co-culture and transfecting plasmid pCMVSV40T/PUR to get a type of immortalized Nucleus Pulposus Cell like cells, which came from Bone Marrow Strom Cells (MSCs).Methods:Primary Nucleus Pulposus Cells (NPCs) and primary bone Marrow Strom Cells (MSCs) were got from 20 rabbits. The NPCs were fluorescence-labeled, and were co-cultured with MSCs(co-culture ratio 1:1).After been co-cultured for 6,9,12,15 and 18 days, the MSCs (fluorescent-negative cells) were selected by Flow Cytometry from the co-culture system. Morphological changes of MSCs were observed, and real-time PCR reaction was performed to assess the mRNA expression of maker genes (Collagen II and Aggrecan) in MSCs.In the co-culture 15 days group, plasmid pCMVSV40T/PUR containing SV40Tag gene was transfected into MSCs, and the MTT assay was carried out to observe the MSCs proliferative ability.Results:Co-cultured with NPCs, MSCs began to express maker genes.15 days later, the mRNA expression of maker genes in MSCs reached the peak level at 0.90 and 0.93.But this peak level was still lower than the mRNA expression of maker genes in primary NPCs (P<0.05).Cell morphology also changed from a spindle into a polygon. After been transfected with plasmid pCMVSV40T/PUR, the MSCs almost had the same proliferative ability with the primary NPCs, and this ability would not decline in the next 20 passages (P<0.05).Conclusion:By co-cultured with NPCs and transfected with SV40Tag gene, we can get a kind of NPC-like cell. This NPC-like cell may be helpful to the cell-based therapy of intervertebral disc degeneration.