| ObjectivePeripheral nerve injury is a clinically common disease.It can be divided into several types according to degree of injury (Sunderland type) and the cause of injury. The traditional repair methods (neurolysis, suture, transplantation and implantation, etc.) are not ideal to the patients of serious injury. Finding the ideal method of treatment has always been the core of research made by traumatic surgery and hand surgery ,etc.In recent years, tissue engineering technology used for the treatment of peripheral nerve injury has been a new object of study for many academics. Schwann cells (SCs) derived from the neural crest is the important structure and function cells of peripheral nervous system.A large number of studies confirm it has an important role in the remyelination and functional recovery of peripheral nerve and spinal cord injury. Schwann cells can't be used as seed cells for clinical application ,because it's derived from human invasive nerve biopsy, and it's limited in vitro proliferation, lacking of access to a large number of daughter cells. Bone marrow mesenchymal stem cells may solve this problem, but it can't generate the real functional Schwann cells. Similarly, as for embryonic stem cells and neural stem cells, they can't be used for clinical application according to their ethical, organizational sources and the rejection caused by allograft. Thus, finding a suitable seed cells for tissue engineering is the key of treatment of peripheral nerve injury.Toma and Miller obtained a precursor cells of multipotent differentiation potential from mouse back skin in 2001, and named it Skin-derived Precursors (SKPs), it can generate neuron, glia, smooth muscle, fat and many other cell types. It has been confirmed that SKPs origins from neural crest during the embryonic development, it's a kind of neural crest-derived precursors. With the development and migration of embryonic, SKPs is mainly found in the hair follicle dermal papilla of adult skin finally. Now, the proliferation, differentiation, culture conditions of SKPs are confirmed, it can proliferate in vitro while maintaining their differentiated characteristics. In the Shivere mice model of peripheral nerve injury, SKPs and the Schwann cells sources from it both can facilitate the myelin process and the recovery of axonal continuity. Thus, SKPs can produce the real functional Schwann cells, combined with it can be easily obtained from the skin of body, and can be used for autologous transplantation, so SKPs could be a new, acceptable source of autologous stem cell transplantation for the treatment of peripheral nerve injury.Although the current study of SKPs has obtained some results, the specific regulatory mechanism of proliferation and differentiation is not clear. The specific mechanism of formation, migration and differentiation of neural crest during the embryonic development has been understood basically now, it has been confirmed that Notch, Wnt, Sox-10 and other signaling pathways and molecules involves in the regulation of neural crest differentiation. Since the SKPs are derived from neural crest, and their biological and differentiated characteristics are very similar with neural crest cells, then we asked whether the regulatory mechanism of differentiation of neural crest cells is similar with it? The research of regulation mechanism of differentiation of the SKPs will help us increase the number of Schwann cell derived from it, and provide suitable seed cells for tissue engineering so as to promote the regeneration and functional recovery of peripheral nerve.We are inspired, so we design the experiment to explore the role of Notch, Wnt and other signaling pathways in the regulation of SKPs differentiation. Methods1. SKPs are derived from the back skin of newborn mice (age of 3d,SPF level), digestion, separation and suspension culture, passaging with the conventional method, nestin is detected by, fibronectin related gene is detedted by RT-PCR.2. Passaged cells are divided into two groups: neuron and Schwann, observe the morphology of cells respectively, immunocytochemical staining detect neuron and Schwann cells marker (NeuN, GFAP), the method of RT-PCR detect neuron and Schwann cells marker (NFM, CNPase) related gene.3. SKPs are sorted by FCM, getting side population cells, and then are induced to neurons and Schwann cell. The cells cultured under differentiation options are collected at different time points in each group , then extracte the total RNA, the RNA is transcripted reversely and labeled in vitro. We detect the differences of gene expression between the two groups with primary SP cells by Microarray. As for the key results, we make further confirmation by method of qRT -PCR.Results1. SKPs derived from mice grow well, form clear speres, and express nestin and Fibronectin.2. SKPs in neuron and schwann group are significantly different in the process of differentiation culture. Cells express NFM and NeuN in neuron group, and they express GFAP and CNPase in schwann group.3. is most obvious between the two groups with primary group after SP. As for the expression of genes continued to9 genes are i upregulated , 34 genes are downregulated in neuron group. of 33 genes are upregulated, 27 genes are downregulated in schwann group. The difference in gene expression is not significant after the cells are cultured for 3 or 7 days. ConclusionsThe gene expression of SKPs is significantly different compared with primary cells during differentiation, and the changes of genes mainly concentrate in the wnt and notch signal pathway. This indicates that these signal pathways have an important regulatory role during the differentiation of SKPs. |
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