The Ability To Form Cartilage Of NPMSC And BMSC In SD Rats

With the incidence of low back pain is increasing, the treatment of this disease has become a medical and social problems. Study found that low back pain is caused by many reasons, including injuries, disc degeneration, disc herniation, spinal stenosis. The disc degeneration is considered to be the main reason for lower back pain. Disc degeneration as follows: reduction of nucleus pulposus cells and extracellular matrix, destruction disc structure. Many factors, including: genes, bad habits, heavy manual labor and other physical disorders and so may lead to disc degeneration.Current treatment of disc degeneration methods: medication, steroid injections, physical therapy, discectomy, spinal fusion and so on. These methods can reduce symptoms, it can not cure and prevent disc degeneration process. Therefore, many new treatments and ways proposed to reverse disc degeneration. These methods focus on repair and regeneration of nucleus pulposus cells. The firs new therapy is molecular therapy, the use of anabolic gene product regulation and degradation pathways to inhibit or reverse disc degeneration. The second new therapy is cell therapy and tissue engineering, through restoration or replacement of degenerated intervertebral disc nucleus pulposus cells to achieve the purpose of treatment. Currently used in the seed cells extracted from the bone marrow, fat, and other tissues out of the umbilical cord stem cells, as well as homology nucleus pulposus cells and chondrocytes.Recently Blanco isolated and cultured some cells from degenerative disc tissue, found that such cells express stem cell surface markers CD44, CD29, CD90, CD73, CD105, CD166, CD106, did not express hematopoietic stem cell markers CD34, CD45, CD14, CD19, and has osteogenic cartilage differentiation without adipogenic differentiation. Compliance with international Society for Cell therapy(the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy, ISCT) standards defined in pluripotent mesenchymal stem cells. NPMSC derived from the nucleus pulposus, compared to other tissue-derived stem cells can adapt to the environment within the intervertebral disc. Whether the nucleus pulposus stem cells and bone marrow stem cells have difference in cell morphology, value-added, ability of osteogenic and adipogenic differentiation, cartilage differentiation capacity, there is no relevant literature.In this study, we extracted the nucleus pulposus stem cells from the SD rat nucleus, and cultured identification. Compare the nucleus of stem cells and bone marrow stem cell morphology, added value and differentiation. Compare the nucleus of stem cells and bone marrow stem cells,what is the difference in the ability of cartilage in vitro conditions. The experiment is divided into three parts:Section 1 The separation culture and identification ofNPMSC Objective:This part of the experiment was isolated and cultured nucleus pulposus stem cell from SD rats, and by cell morphology, stem cell genes, stem cell surface markers, and osteogenic, adipogenic, chondrogenic differentiation were identified. Materials and methods: 1. Experimental AnimalsHealthy male SD rats(weight 250 ± 10 g by the Experimental Animal Center of Shanxi Medical University). 2. Experimental Methods 2.1 NPMSC IsolationApplication microscope isolated nucleus pulposus from each rat tail SD disc under sterile conditions. After 0.2% type II collagenase and 0.25% trypsin treatment, containing 20% fetal calf serum and penicillin-streptomycin in DMEM / F12 culture medium.2.2 NPMSC cell morphologyNPMSC culture to the third generation cell morphology. 2.3 PCR detection of genetic testing NPMSC stem cellsCollected the third generation NPMSC applications Trizol extraction of total RNA. PCR detection of Nanog, Sox2, Oct-4 gene expression in stem cells. 2.4 PCR detection NPMSC stem cell surface markerPCR to detect the expression of stem cell surface markers.CD44, CD105, CD73, CD90, CD45, CD34, CD14, HLA-DR 2.5 NPMSC osteogenic, adipogenic, chondrogenic differentiationNPMSC in vitro conditions to differentiate into bone, fat, cartilage. Observe the ability of differentiation. Results: 1. NPMSCs can be isolated and cultured from the nucleus pulposu of SD rats. 2. The third generation NPMSC shape remained the same as the spindle-like fusiform. 3. NPMSC stem cell gene expression of Nanog, Sox2, Oct-4. 4. NPMSC expressed stem cell surface markers CD44, CD105, CD73, CD90, did not express hematopoietic stem cell surface markers CD45, CD34, CD14, HLA-DR. 5. NPMSC have the ability of osteogenic, adipogenic, chondrogenic differentiation Summary:NPMSC can be isolated cultured from the nucleus pulposus of SD rats,long spindle-like spindle cells express stem cell gene Nanog, Sox2, Oct-4, the expression of stem cell surface markers CD44, CD105, CD73, CD90, did not express hematopoietic stem cell surface markers matter CD45, CD34, CD14, HLA-DR, has osteogenic, adipogenic, chondrogenic differentiation capacity.Section 2 Compare the differentiation capacity of NPMSC and BMSC Objective:Isolation and culture NPMSC and BMSC from SD rats, comparison the two stem cell of morphology, cell proliferation ability, gene expression of stem cells, stem cell surface marker expression, osteogenic、adipogenic,、chondrogenic differentiation capacity, as well as induced osteogenic, adipogenic, chondrogenic gene expression. Materials and methods: 1. Experimental AnimalsHealthy male SD rats(weight 250 ± 10 g by the Experimental Animal Center of Shanxi Medical University). 2. Experimental Methods 2.1 NPMSC and BMSC isolation and cultureNPMSC and BMSC were isolated and cultured from SD rat nucleus pulposus and bone marrow. 2.2 NPMSC and BMSC cell morphologyNPMSC, BMSC cultured to the third generation of cell and detected morphology. 2.3 NPMSC BMSC ability of cell proliferationApplication of CCK-8 were detected by the third generation NPMSC and BMSC cell proliferation ability in 0, 1, 3, 5, 7, 9. 2.4 PCR detection NPMSC and BMSC stem cell geneticCollect the third generation NPMSC, BMSC application Trizol extraction of total RNA. PCR detection of Nanog, Sox2, Oct-4 gene expression. 2.5 PCR detection NPMSC and BMSC stem cell surface marker detectionPCR to detect the expression of the third generation NPMSC and BMSC in stem cell surface markers(CD44, CD105, CD73, CD90, CD45, CD34, CD14, HLA-DR). 2.6 NPMSC and BMSC osteogenic, adipogenic, chondrogenic differentiationNPMSC, BMSC in vitro conditions to osteogenic, adipogenic, chondrogenic differentiation. Observe NPMSC, multi-directional differentiation compare with BMSC. 2.7 Osteogenic, adipogenic, chondrogenic gene expressionPCR detection Osteogenic, adipogenic, chondrogenic gene expression of NPMSC and BMSC. Results: 1. NPMSC and BMSC s were isolated and cultured from SD rats. 2. The third generation morphology of NPMSC and BMSC are spindle-like long spindle consistently.3. The third generation NPMSC and BMSC are similar in value-added capabilities. 4. NPMSC and BMSC were expressed stem cell gene Nanog, Sox2, Oct-4. 5. NPMSC and BMSC expressing stem cell surface markers CD44, CD105, CD73, CD90, did not express hematopoietic stem cell surface markers CD45, CD34, CD14, HLA-DR. 6. NPMSC and BMSC have the ability of osteogenic, adipogenic, chondrogenic differentiation. 7. NPMSC and BMSC expressed osteogenic, adipogenic, chondrogenic genes after differentiation. Summary:NPMSC isolated from cultured from SD nucleus pulposus tissue and BMSC from long bone marrow,compare the morphology, ability to add value consistent, both expressing stem cell gene, expression of stem cell surface markers, did not express hematopoietic stem cell surface markers, with the ability of osteogenic, adipogenic, chondrogenic differentiation, after induced expressing osteogenic, adipogenic, chondrogenic genes.Section 3 chondrogenic differentiation capacity of NPMSC and BMSC Objective:PCR, RT-PCR and Western-blotting were used to detect collagen II, proteoglycans, SOX9 gene and protein expression after differentiation. Comparison of two stem cell differences in the ability of chondrogenic differentiation. Materials and methods: 1. Experimental AnimalsHealthy male SD rats(weight 250 ± 10 g by the Experimental Animal Center of Shanxi Medical University). 2. Experimental Methods 2.1 NPMSC and BMSC differentiation into chondrocytes in vitro conditionsThree generations of stem cells 2.5 × 105 centrifuge, add 1ml fresh complete chondrogenic solution(1ml incomplete chondrogenic liquid + 10μTGF-?3), cells were collected after 21 d of cartilage induced. 2.2 PCR detected the expression of type II collagen, proteoglycans, SOX9 geneTotal RNA was extracted after NPMSC and BMSC induced, using PCR to detect gene expression of type II collagen, proteoglycans, SOX9. 2.3 RT-PCR to detect the expression of type II collagen, proteoglycans, SOX9 geneTotal RNA was extracted after NPMSC and BMSC induced, application of RT-PCR detected the gene expression of type II collagen, proteoglycan, SOX9. 2.4 Western-blotting to detect the expression of type II collagen, proteoglycans, SOX9 proteinTotal cellular protein were extracted induction of NPMSC/BMSC and before induction, the application of Western-blotting quantitative detection protein of type II collagen, proteoglycans, SOX9. Results: 1. NPMSC and BMSC can be differentiated cartilage in vitro conditions. 2. NPMSC and BMSC into PCR detection after the induction of cartilage type II collagen and proteoglycans, SOX9 gene expression were increased, no difference in expression between the two. 3. After chondrogenic differentiation of NPMSC and BMSC, using RT-PCR detection gene expression of type II collagen, proteoglycans, SOX9,which were increased, there was no difference in expression between the two. 4. NPMSC and BMSC after chondrogenic differentiation,Western-blotting quantitative detection protein expression of type II collagen, proteoglycans, SOX9 were increased, while there was no difference between the two. Summary:After NPMSC and BMSC induced into cartilage, using PCR, RT-PCR, Western-blotting detected gene and protein expression of type II collagen, proteoglycans, SOX9,that was significantly higher than before induction. There was no significant difference between NPMSC and BMSC. Conclusion:In a word, in this study we confirmed that NPMSC with characteristics of stem cells can be isolated and cultured form nucleus pulposus tissues of intervertebral disc of SD rats, the chondrogenic ability of NPMSC and BMSC was similar under induction in vitro. This could provide a new seed cells for tissue engineering.