| In resent years, stem cell transplantation has been considered as one of the most promising therapy strategies in repairing nervous injury. In this field, the stem cells which are concerned and studied widely include embryonic stem cells,neural stem cells,bone marrow-derived mesenchymal stem cells and mesenchymal stem cells from umbilical cord blood and so on. Apart from bone marrow-derived mesenchymal stem cells, the other types of stem cells need to be acquired from allosome, so the application for them will involve the problem of ethics and immune rejection. While the subject will be painfull in the process for acquiring bone marrow-derived mesenchymal stem cells. So it is necessary to search and research a better source for stem cells.Many researches have indicated that the phenotype of mesenchymal stem cells from umbilical cord blood is very similar with that of bone marrow-derived mesenchymal stem cells(BMSCs), and possesses the ability of multiple differentiation and proliferation. In terms of developmental biology, the umbilical cord blood is actually foetal blood, so the stem cells in umbilical cord blood come from the stem cells in bone marrow. That means that there will be much multi-potential stem cells in the body until the birth. Considering that, our study was to explore the existance of these stem cells in peripheral blood after birth, and establish the method for the isolation and culturing of PBMSCs.Schwann cells, which can secret many neurotrophic factors to promote the nerve regeneration, are usually used as the repairement of central and peripheral nerve injury. But the source of Schwann cells will be limited if the traditional method of acquiring source of Schwann cells from peripheral nerve is used. In resent years, many researchers have tried to acquire the Schwann cells by the method of inducing differentiation from stem cells. Therefore, we explore the method of inducing differentiation into Schwann cells in vitro on the basis of acquiring PBMSCs for their further application in repairing nerve injury.This research is divided into three parts:The first part:Extracted blood from SD rat to separate and culture PBMSCs,detected and identified the surface antigen of PBMSCs with flow cytometry and immunocytochemical method, and the flow cytometry showed the positive rates of CD29,CD90,CD45,CDllb,CD106 and CD49d in cultured PBMSCs were 99.96%,71.22%,46.62%,19.97%,10.76% and 5.46% respectively. The immunocytochemical method revealed that CD34 was negative in PBMSCs. And detected the positive rate of BrdU with immunocytochemical method after BrdU involed 4h. The positive rate of BrdU in PBMSCs was (34.1±4.3)%. Based on the above fact, the stem cell separated from peripheral blood had the characteristics of mesenchymal stem cell.The second part:Detected the expression of S-100 and P75 with immunocytochemical method after PBMSCs were induced to differentiate into Schwann cells through the following steps:β-mercaptoethanol, all-trans retinoic acid and compound induce medium (1% N2,50ng/ml heregulin,5μM forskolin,10ng/ml bFGF and 5ng/ml PDGF. The result showed the positive rates of S-100 and P75 were (75.2±4.1)% and (78.9±4.6)% respectively. This indicated that PBMSCs could be induced to differentiate into Schwann cells under certain conditions.The third part:Transplanted PBMSCs with GFP positive into sciatic nerve after the nerve was exposed. After 6 weeks living, drawed materials, sliced and performed the morphology observation. The result revealed that the Transplanted PBMSCs with GFP positive in sciatic nerve had differentiated into Schwann cells, presenting GFP/S-100 and GFP/P75 double positive. The staining result for GFP/NF200 and GFP/MBP displayed that the differentiated Schwann cells from PBMSCs had formed medullary sheath circled the axon. So the result demonstrated that PBMSCs could differentiate into s in vivo, and form medullary sheath. |
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