Experimental Study Of Schwann Cells Cultured Neonatal Rat Sciatic Nerve

Objective:1. Exploring the separation cultured and conditions required for passed of schwann cells from neonatal rat,in order to provide seeds of schwann cells for further transgenic and transplantation.2.Observating the proliferation and secretion of Schwann cell in the effects of different concentrations of brain-derived growth factor cultured environment.Exploring the effects of brain-derived growth factor (BDNF) to the proliferation and experimental study of Schwann cells secreted in vitro.Methods:1. Borned5-7days of eight neonatal rats were sacrificed, separating its bilateral sciatic nerves, at low temperature in sterile conditions.Double enzyme digestion method into a single cell suspension, adding10%FBS high glucose medium, placed in pre-fitted with polylysine culture flask, using30minutes differential preliminary removal of adherent fibroblasts, the resulting cell suspension was placed in37℃,5%carbon dioxide incubator。24hours later, the medium was added to a final concentration of10-5/L cytarabine further purified Schwann cells. After24hours and replaced with medium containing foskilin bovine pituitary extract in the culture. Daily observation, recording growth conditions Schwann cell growth curve. Growing well Schwann cells were passaged using immunofluorescence techniques to identify them.2.Taking5-7days pregnant neonatal rat sciatic’s schwann cells of sciatic nerves,which were placed in brain-derived neurotrophic factor and10%FBS and DMEM medium, resulting to a final concentration of BDNF medium0,10,20,50,100ng/ml.Placing the schwann cells in the five groups and culturing for1week,during the time, observing by MTT assay and comparing the growth and proliferation of schwann cells. Using ELISA to detect the concentration of BDNF do the effect to the situationg of Schwann cells secrete nerve growth factor (nerve growth factor, NGF), ciliary ganglion neurotrophic factor (ciliary neurotrophic factor receptor, CNTF).Results:1.Schwann cells isolated from neonatal rat sciatic nerve. Inverted microscope to see24after the start of the cultured cells adherent, adherent cells and more small, round, bright, strong refraction, do not move with the flow of the liquid, a small amount of cells were bipolar cord-shaped or triangular, round or into the nucleus oval, more suspension cells.7-8days later, after the medium was changed three times. All adherent cell growth, cell shape mostly bipolar cord, a few cells triangular protrusions are connected between adjacent cells, or into bundles arranged parallel to each other. After the cells were passaged bipolar cable lines, adherent growth. By immunofluorescence staining S-100identification, bipolar Schwann cells were mostly arranged parallel to each other, the highest purity of80%.2.BDNF final concentration of50ng/ml,100ng/ml Schwann cell viability was significantly higher than the final concentration of BDNF proliferation activity Ong/ml Schwann cells, and the difference was statistically significant (P<0.05), and BDNF final concentration of10,20ng/ml final concentration Ong/ml group compared to the group, although there are differences in the vitality of Schwann cells, but no significant difference (P>0.05). In addition, Schwann cells can secrete NGF, CNTF, BDNF when the final concentration of50ng/ml,100ng/ml final concentration Ong/ml with BDNF compared Schwann cell NGF, CNTF expression levels were significantly increased, and the difference was statistically significance (P<0.05). And when the final concentration of50ng/ml BDNF, the Schwann cell NGF, CNTF expression levels were significantly highest, but the difference was not statistically significant100ng/ml.(P>0.05).Conclusions:1. Using double enzymatic to digeste nerve tissue into single cells, adding cytarabine inhibit fibroblast growth, then adding foskilin to promote schwann cell proliferation is a more efficient and economical method,and it can successfully obtain the schwann cells from neonatal rat sciatic nerve. After immunofluorescence staining, the specific expression of Schwann cells purified S-100ratio of up to80%,so it can be used to further transgenic experiments or transplant.2.Brain-derived neurotrophic factor have a certain role in promoting proliferation and secretiont on cultured Schwann cells in vitro.