| Background and Purpose：Bone mesenchymal stem cells (BMSCs) andadipose derived stem cells have the basic characteristics of stem cells, whichare the ability of self-renewing and could be differentiated into other cells.Intervertebral disc degeneration is mainly characterized by the degradation ofextracellular matrix, reduction of nucleus pulposus cells and functionaldecline. The purpose of this study is thus to observe the cell morphology,measure the prolife time of different cells and count the cells number.Homologous human bone mesenchymal stem cells (BMSCs), adipose derivedstem cells (ADSCs), degenerative nucleus pulposus cells (NPCs) will beharvested with informed consent from bone marrow, adipose and nucleuspulposus of patients undergoing lumbar spine surgery.Methods: The bone marrow, adipose tissue and nucleus pulposus tissuewere harvested from a same patient, after cut into pieces by eye scissors, thenucleus pulposus tissue and adipose tissue were respectively treated withcollagenase typeⅡand typeⅠdigestion, the bone mesenchymal stem cellswere separated by using density gradient centrifugation method. after that，thestem cells and nucleus pulposus were cultured in DMEM/F12medium，whichsupplemented with felal bovine serum. And then to observe the cellmorphology, measure the prolife time of different cells and count the cellsnumber.Results: BMSCs and ADSCs were isolated from bone marrow andadipose tissue and expanded as primary cultures.24hours later, the cellsbegan to attach to culture flask, and the cells spread out and were shown asfibroblast-like morphology gradually after1week. The cells could be passagewhen fused to80%~90%2weeks later, the cells became to be fusiform shape,swirly and evenly distributed. The nucleus nulposus began to attach to culture flask after4~5days, morphology of cells was long fusiform shape, and couldbe passage10days later.Conclusions: In this study, we isolated BMSCs and ADSCs from humanbone marrow and adipose tissue. The NPCs could also be attached to cultureflask. We have observed that the BMSCs and ADSCs displayed thecharacteristics of mesenchymal stem cells, but ADSCs have a big storage andwith a smaller trauma when harvest compared with BMSCs. The two kinds ofstem cells both might be co-cultured with degenerative NPCs. Background and Purpose: The degenerative disc disease (DDD) wasthe main reason for low back pain (LBP) and neck and shouder pain. Theincidence of spinal degenerative disease increased significantly commonlyfollowing the extension of life expectancy. For another expect, the LBP has ayounger trend because of fast life rhythm and big working pressure. However,the current clinical therapy for degenerative disc disease long-term effect isnot ideal. Committed to repair rather than simple excision of degeneration ofintervertebral disc treatment is an innovative train of thought. Using stem celltransplantation to repair degenerative intervertebral disc gradually becomeimportant development direction in the field of spine surgery, may bring tothe treatment of intervertebral disc degeneration related disease progress.Although many kinds of stem cells in the basic research shows goodprospects for repair of intervertebral disc degeneration, but it is lack ofcontrast experiment, still can’t confirm what kind of stem cells suitable forintervertebral disc repair one of the best sources of seed cells. In this research,we aquared bone marrow mesenchymal stem cells，adipose tissue-derivedstromal cells and nucleus pulposus（NPCs） from the same patient whentaking spinal operation.Then the two kinds of stem cells and homologoushuman degenerative nucleus pulposus cells for non-contact cocultivation,discuss which kind of mesenchymal stem cell is more suitable fordegenerative disc disease biology treatment?Methods: In the supervision of the hospital ethics committee and underthe premise of fully respecting patient informed consent right, we gather bonemarrow, fat tissue and nucleus pulposus tissue from the same patient whentaking spinal operation. Gathering human bone marrow, fat and nucleus pulposus tissue.After isolation and proliferation, bone marrow mesenchymalstem cells (MSCs) and adipose stem cells (ADSCs) respectively coculture(cells’ ratio is50:50) with homologous degenerative nucleus pulposus cells inTranwell six-well paltes for1week. The transwell six-orifice plate insertedinto the layer is0.4microns in diameter at the bottom of the semi-permeablemembrane; The bone marrow mesenchymal stem cells and adiposemesenchymal stem cells were inoculated in six orifice plate bottom, nucleuspulposus cells inoculated in six-orifice transwell semi-permeable membraneat the bottom.. With ADSCs cultivation of NPCs for group A, and BMSCscultivation of NPCs for group B, nucleus pulposus cells cultured alone servedas control. One week later, when the cDNA were obtained from reversetranscription, relative gene expressions of type II collagen，SOX-9andaggrecan were determined by using the Real-Time PCR and normalized bythe housekeeping gene of GAPDH. Nucleus pulposus cultured alone served ascontrols. The2-ΔΔCtmethod was then used to calculate the value of the relativeexpression for each target gene.Results: The Real-Time PCR results showed that compared with thecontrol group, the nucleus pulposus cells of group A and B had a significantincrease of Ⅱ collagen protein, proteoglycan and SOX-9gene expression(P <0.05); The relative gene expression of Ⅱ collagen protein, proteoglycanand SOX–9in group A respectively are3.49±0.55,3.88±2.11and2.41±0.91,while7.60±1.89,6.26±2.96and4.55±1.88in group B, the twogroups have a significant difference(P <0.05).Conclusions: Bone marrow mesenchymal stem cells and adiposemesenchymal stem cells can both stimulate and activate the degenerativenucleus pulposus cells under the condition of noncontact co-culture, and witha significantly increase of collagen type Ⅱ, polysaccharide and SOX-9indegenerative nucleus pulposus cells;Bone marrow mesenchymal stem cellshave a stronger activation effect for degenerative nucleus pulposus cells, itmay be more suitable for degenerative disc diseases’ biological treatment. |
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