| intervertebral disc degeneration is a serious impact on the lives of common diseases, as lumbar back pain is one of the main reasons, its degeneration is largely due to internal disc nucleus pulposus cells reduce and extracellular matrix components of change. In recent years disc tissue engineering, use seed cell transplantation of tissue damage and to promote the recovery of the treatment method becomes the current medical field most. In many other seed cells, adipose tissue of adipose-derived stromal cells with content, location and superficial easy sources of proliferation, is a lot of differentiation potential of adult stem cells, many experiments have proved adipose-derived mesenchymal stem cells can differentiate into disc-like cartilage cells. Cell culture techniques of micromass in tissue engineering indicates that, in the center of the cell micromass due to hypoxia and low nutritional status, and the nucleus pulposus cell survival similar, more conducive to seed cell to the nucleus pulposus-like cartilage cell differentiation. Based on the induction of adipose-derived stromal cells into nucleus pulposus-like cartilage cell differentiation of various studies, the lab is designed by and nucleus pulposus cells and cell factor two comparisons, induced by way of exploration in vitro cell differentiation, the best way to future seed cell transplantation in the treatment of degenerative disc disease provide experimental basis.Method1.Get New Zealand rabbits groin, armpit fat, 0.3% of the type II collagen enzyme collagenase solution under shear and Digest 40 min (37°c), the digestive fluid filtration, centrifugal gets adipose-derived stromal cells. Get New Zealand rabbits whole spine, open fiber ring for Nucleus Pulposus Tissue, 0.3% of the type II collagen enzyme collagenase solution under shear and digestion 30 min (37°c), the digestive fluid filtration, centrifugal Gets the nucleus pulposus cells. The collected fat mesenchymal stem cells and cell nucleus pulposus with DMEM/F12 (1: 1) medium monolayer culture respectively.2. The adipose-derived mesenchymal stem cells culture to 3 generations are qualified, press 5×106nucleus pulposus cells and adipose-derived mesenchymal stem cells in Eppendorf tube centrifugal, 3-4 days made into cells micromass.3. The adipose-derived mesenchymal stem cells micromass into cultivation, Transwell lower respectively by 5×106 nucleus pulposus cells micromass and TGF-β1/I G F-1 induces; to before induction, induced 7, 14 days, observation cell shape changes, and through RT-PCR on collagen and proteoglycan.ResultsIn 7, 14-day, two groups of adipose-derived mesenchymal stem cells of size, shape and delusion, RT-PCR detection results display a 7-day TGF-β1/I G F-1 group and nucleus pulposus induction group has type II collagen and proteoglycan mRNA expression, but the TGF-β1/I G F-1 group expression stronger; induced 14-day induction group nucleus pulposus collagen and proteoglycan mRNA obviously increase, TGF-βsuperior but 1/I G F-1 group.ConclunsionIn vitro NP and TGF-β1/I G F-1 lead to the ADSCs is promoting its role to the NPCs differentiation, TGF-β1, IGF-1 for normal nucleus pulposus cell secretion important factor, the result of NPCs to ADSCs cytokine induction effect between the subject, yet exists for the promotion and proliferation of differentiation, as better differentiation to the nucleus pulposus is still other induced conditions exist.Nucleus pulposus cells and adipose-derived mesenchymal stem cells from a three-dimensional cultivating cytokine-induced economic and effective, not only can better promote seed cells into purpose cell differentiation, and can simulate intradiscal environment to facilitate future in vivo.
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