Mutual Effects Between The Neuroepithelial Stem Cells And Schwann Cells Co-Cultured Together

Background:Schwann cell is the mainly glial cell in the peripheral neural system and promotes nerve regeneration and recovery of function. It is seen as the candidate cell in the cell transplantation. While it is difficult to preserve the autoallergic Scs proliferate massively and steady characters, moreover it needs two times surgery; the allograft may cause the immunological rejection in the host. So it is necessity to look for other cells more fit the transplantation continuously. The neural stem cells was different to the Scs like cells before transplanted into the PNS, these cells could improve the micro-environment of regeneration, promote the axons regrowth ,the neurons differentiated from the NSC could support the target organs which were controlled by the damaged nerves and prevent these tissue atrophying. This phenomenon made people think the NSC may have a wide potentiality treating the damaged nerves. The neuroepithelial stem cells may different into more neurons and may be considered superior to repair the neural pathway. There are some reports on the survival, differentiation and effects about the NSC. But the mechanism and the inducing factors of the neuroepithelial stem cells and the effects between the Scs and neuroepithelial stem cells are known little.Object:The regeneration of the damaged nerves is along the focus in the fields of traumatic surgery and neuroscience. The applying of Scs is limited by insufficiencies though it is still as the preferred candidate in the tissue engineering fields. With the study deepening in stem cells, the neural stem cells show its huge potential in the treatment of PNS not just only in the CNS disease. So search for how to realize a advantageous environment for the regeneration of damaged peripheral nerves, how to gain sufficient Scs for the transplantation ,how promote the survival of the NSC in transplant areas and the neurons amount differentiated from the NSC, would give more efficient strategies for treating the diseases in the PNS. On account of the above, we design this experiment: co-culturing the neuroepithelial stem cells and Scs in vitro, then observing the survive and differentiation of the neuroepithelial stem cells, the proliferation and expressing MBP of Schwann cells. The aim is to discuss the optimum conditions of gaining sufficient Schwann cells and the neurons at the same time and to provide theoretic and experimental evidence for the treating the diseases in the PNS through cells-transplant.Methods:In this study we isolate and purify the Schwann cells from the sciatic nerves and median nerves of neonatal Wistar rats and the neuroepithelial stem cells from the neural tube of embryo Wistar rats (E11.5d).1. To observe the morphology by the inverted phase contrast microscope and record the changes by the system of image collection.2. Planting the neuroepithelial stem cells on the coverslips coated by the polylysine, the control group was culturing the neuroepithelial stem cells only. A week later, fixing the cells and showing the dealing with MAP-2,GFAP positive cells by immunocytochemistry. Collecting the image and anglicizing the ratio of the MAP-2 to GFAP positive cells by the soft of IPP.3. Studying the Schwann cells activity of different groups by MTT assay.4.Immunocytochemistry: to show the expression of the MBP in the system of co-culturing the neuroepithelial stem cells and the Schwann cells.5. SDS-PAGE and Western-blot: Extracting the total protein of co-culturing the neuroepithelial stem cells and the Schwann cells, detecting the MBP by SDS-PAGE and Western-blot. Result:1.The neruoepithelial stem cells grow suspended and form the neurospheres by division growth. With persistent culture, the amount of the cells in the neurosphere is increased rapidly and hundreds of cells construct a sphere in the 5～6 days. The spheres were nestin positive and MAP2 and GFAP positive cells were around the spheres by immunocytochemistry.Primary Schwann cells planted just now were round, suspending in the medium. More cells attached on the wall after 24 hours .The cells were comparative small with a dynamic scene of three-dimension and satiation and more were shuttle-shape with two slender dendrites. These cells were S-100 positive.In the co-culture system the neurospheres were attached quicker than the control group. After 24 hours more spheres attached steadily and after 36 hours the cells emigrated from the neurospheres. The emigrating cells were round and full and two to three branches were observed. These cells were like the mature neurons morphologically. The Schwann cells were not marked changes early. When the neuritis stretched out from the neurospheres long enough, they were arranged sarciniform and the double-track formation were observed in the bright field of the microscopic. In the control groups the neurospheres grow badly and few attached the wall or attached unstable and no living cells were observed a week later.2. The ratio of the MAP-2 to GFAP positive cells in the experimental groups were 1.53:1 and with the serum up the ratio were reduced. There were no positive cells in the control groups.3.MTT shows that the conditional medium promote the survive and proliferation of the Schwann cells than the control group (P<0.01).4.Part of the neurites were MBP positive along the neurites by immunocytochemistry. This demonstrates that the Schwann cells could be induced to differentiate towards the myelined Schwann cells by the neurons derived from the neruoepithelial stem cells.5. MBP was detected in the group of co-culturing neuroepithelial stem cells and the Schwann cells but the content was less than the nerve tissue. But MBP were not detected in the Schwann cells groups.Conclusion:Co-culturing the neuroepithelial stem cells and Schwann cells is fit for the neuroepithelial stem cells surviving and differentiating into neurons and for the Schwann cells' proliferation, the neurons are matured enough to induce the Schwann cells differentiating to myelinating Schwann cells and expressing MBP.