| Background For many years,the repair of nerve injury has been a difficult medical problem.So far,nerve auto-transplantation has been still the "gold standard" for the treatment of nerve broken ends,but this method has the shortcomings of insufficient donors.For this reason,people are looking for various alternative materials,such as chitosan,silk protein,fiber catheter and so on.Although these materials have made some progress in the recovery of nerve function,they are still unsatisfactory.Schwann cells,the myelin sheath cells encapsulating axons,play a major role in repairing axons,supporting and guiding axon regeneration,secreting various cytokines to support axon regeneration and phagocytose cell debris produced during cellular inflammation.Previous studies have shown that mesenchymal stem cells can repair the crush injury of sciatic nerves in SD rats and improve the nerve function.Embryonic stem cells are omnipotent stem cells which have the potential to differentiate into all directions.They can differentiate into specific cell groups by appropriate microenvironment.Human embryonic stem cells have broad clinical application prospects and significance.At the same time,human embryonic stem cells and their induced products can avoid immune rejection caused by xenotransplantation in further clinical applications.Human embryonic stem cell-derived neural stem cells in the repair of nerve injury may provide a variety of nutritional factors and signals to help repair,which is a potential tissue functional material for nerve repair.Objectives To explore the differentiation of human embryonic stem cell-derived neural stem cells and the biological effects of neural stem cell conditioned medium on rat sciatic nerve Schwann cells,and provide new ideas for the repair of peripheral nerve injury.Methods(1)Identification of neural stem cells derived from human embryonic stem cells and culture of neurospheres.(2)Identification of neural stem cells by flow cytometry and immunofluorescence.(3)Isolation and purification of primary Schwann cells from SD rats and identification by immunofluorescence.(4)Preparation of conditioned medium derived from human embryonic stem cells neurospheres.(5)The proliferation of Schwann cells were detected by CCK-8 assay.(6)The migration of Schwann cells were detected by wound healing assay and transwell assay.(7)The gene expression of Schwann cells were detected by RT-q PCR.Results(1)Human embryonic stem cells showed typical rosette structure after induction and differentiation.The positive rate of Nestin and ? III Tubulin was 98.14%.(2)The expression of S100 and MBP in rat Schwann cells was 99.0% and 94.6%respectively after purified.(3)50% h ESCN-CM could promote the proliferation of Schwann cells,while 100%h ESCN-CM could inhibit the proliferation of Schwann cells.50% h ESCN-CM and 100% h ESCN-CM could promote the proliferation of RSC96.(4)h ESCN-CM can promote migration of Schwann cells.(5)The expression of NT3,VEGF,b FGF and NGF was increased after treatment 4hrs,and the change of IGF-1 was not obvious.Conclusions(1)The differentiation of human embryonic stem cells into neural stem cells was successfully induced in vitro.(2)Human embryonic stem cell-derived neurospheres conditioned medium can promote the proliferation and migration of Schwann cells at appropriate concentration.(3)Human embryonic stem cell-derived neural stem cell conditioned medium can promote the expression of NT3,VEGF,b FGF and NGF genes in Schwann cells at appropriate concentration. |
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