| Low back pain is one of the most common reasons for seeking medical advices and major reasons of work-related disabilities. The most common cause of low back pain is degenerative disc disease (DDD). At present, Treatments for the DDD patients can be classified into two categories, the conservative treatment and surgical intervention. Conservative treatment can temporarily alleviate the clinical symptoms, but with the recurrent of clinical symptoms, so the long-term efficacy is not ideal. The operation treatment of intervertebral disc discectomy and fusion can completely solve the degeneration problem of the responsibility segments, but can be applied only to patients with advanced degeneration. And there is a likelihood of postoperative complications. For example, due to the interference to the biomechanical characteristics of the vertebral body by the spine operation, intervertebral fusion may accelerate disc degeneration of adjacent segment and lead to the occurrence of adjacent vertebral disease (ASD). what’s more, the biomechanical function of intervertebral disc is so complex that the clinical applications of new technologies, such as artificial intervertebral disc, pith nucleus replacement, and allogeneic disc replacement technology is also difficult to completely simulate and alternative the normal function of intervertebral disc. So new operation technologies also exist some clinical complications and long-term problems. In contrast, the stem cell therapy to intervertebral disc is an ideal way to repair the disc degeneration, especially for early stage of intervertebral disc degeneration. Research shows that the etiology of intervertebral disc degeneration is due to the long-term stress and accumulation damage of intervertebral disc, leading to intervertebral disc matrix metalloproteinase and its inhibitor imbalance, abnormal expression of inflammatory cytokines regulate factors. Finally, lead to the activity and cell numbers of the intervertebral disc decreased, and finally caused the disc matrix synthesis and decomposition imbalance. Stem cells treatment for intervertebral disc degeneration is that transplantation of stem cells and the differentiatied it into nucleus pulposus-like cells to increase matrix of intervertebral disc cells, to delay or finally reversing intervertebral disc degeneration.Mesenchymal stem cells (MSCs) is a kind of precursor cells having self-replication and differentiation ability. It can be induced into a variety of functional cells such as bone cells, cartilage cells, muscle cells, fat cells, blood vessel cells, nerve cells, tendon cells, ligament cells in a certain condition. So it is an ideal source of transplanted cells of intervertebral disc. At present, treatment related stem cells of the degeneration of intervertebral disc are the bone marrow or adipose derived mesenchymal stem cells. But the exogenous cells may have deficiency, because exogenous cells may unable to withstand the intradiscal high-pressure, low nutrition, high osmotic pressure environment.Whether the in the adult nucleus pulposus existed nucleus pulposus mesenchymal stem cells, this question is not clear. But there are some evidences of its existence.1. Hohaus reported the application of adult degenerative nucleus pulposus tissue cultured in vitro after replantation, found that the height of intervertebral space increased and nucleus pulposus tissue regeneration. The repair effect may be derived from the nucleus pulposus mesenchymal stem cells.2. Recently, Risbud isolated and confirmed the intervertebral disc containing skeletal progenitor cells from mild degeneration of intervertebral disc tissue (or bone marrow mesenchymal stem cells).3. Blanco also found the presence of endogenous stem cells in intervertebral disc, and named it the nucleus pulposus mesenchymal stem cells (NP-MSC) This study is designed to isolated nucleus pulposus mesenchymal stem cells from normal and degenerated human nucleus pulposus separately. To observe the cell morphology, detect cell immunophenotype and whether it having the potential of multi-directional differentiation, determine whether the isolated cells meet the criterion with mesenchymal stem cells. And compare the normal and degenerative NP-MSC in cell viability and stem cell related gene expression. Ultimately provide an ideal source of seed cells of stem cell trement for degenerative disc disease.Objective:1. Isolation and culture of mesenchymal cells from nucleus pulposus and identified the ability of multi-directional differentiation and surface marker, to confirm if both the normal and degenerative nucleus pulposus tissues are containing mesenchymal stem cells.2. To found whether there are differences in cell proliferation, cell activity and stem cell related genes expression in normal and degenerative derived NP-MSC.Methods:Non-degenerative nucleus pulposus were obtained from spine surgical operation (lumbar burst fracture in4cases,2cases of congenital scoliosis) and degenerative nucleus pulposus were all obtained from6cases of the patients with lumbar disc herniation who need a removal of nucleus pulposus operation. And the nucleus pulposus tissue wre sterile storage in D-Hanks solution,2-6℃preservation.The human nucleus pulposus mesenchymal stem cells were isolated using the the standard MSC culture medium (Cyagen Biosciences, Guangzhou, China) as suitable for MSC culture from6non-degenerative patients of scoliosis or burst fracture and6patient with degenerative intervertebral disc separately. And then two cases of cells were selected from each group, respectively. And analyzed immunophenotype (CD90/CD105/CD73/CD45/CD34andHLA-DR) and multilineage differentiation potential under the criteria to define MSC, which is stated by the International Society for Cellular Therapry (ISCT). The second passage cells from each group was analyzed cell activity using Cell Counting Kit-8(CCK-8/WST-8). Both the total RNA of non-degenerative and degenerative group were extracted, then the Real-Time PCR technology were used to detect the relative expression of stem cell-related genes Oct4and Nanog.Results:NP-MSCs were isolated from both the non-degenerative and degenerative nucleus pulposus. After attachment, the cells gradually spread out and were shown as fibroblast-like morphology. When cultured for3weeks can reach90%cell fusion. After passage, cells proliferation significantly faster, spread to the6generation proliferation ability was not decreased. According to the immunophenotypic analysis of flow cytometry, the NP-MSCs expressed CD73, CD105, CD90, but did not express CD34, CD45and HLA-DR. Detection of cell activity by CCK-8showed the NP-MSCs derived from Non-degenerative nucleus pulposus stronger cell activity than the degeneration ones. Cell viability assay by CCK-8showed that cell activity in normal group was stronger than the degeneration group on the detection point of5,7,9,11,13days, there was significant difference between the two groups (P<0.05). The Real-Time PCR results showed that "Sternness maintenance" related gene Oct4and Nanog expression in normal group were4.63±1.17,7.36±1.19times than degeneration group. The "Sternness maintenance" related gene expression in normal group were significantly higher than the degeneration group (P<0.05).ConclusionBoth the non-degenerative and degenerative NP contain nucleus pulposus mesenehymal stem cells(NP-MSC). Moreover, the non-degenerative NP-MSC showed stronger ability of cell metabolic activity and stem cell-related genes expression. Cell activity and the "Sternness maintenance" related genes expression of the NP-MSC decrease with the degeneration of intervertebral disc. |
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