Direct Reprogramming Of Fibroblasts To Nerve Cells

Objective:In this experiment,first we use ST3205 virus particles infected fibroblasts,and select GFP+ cells with puromycin,subsequently we added nerve cell medium to promote cell differentiation.Then we must observe and record the morphological changes of cells under a microscope.After that we useimmunfluorescence,confocal laser to detect the expression of MAP-2,?-tubulin,MBP,GFAPand so on.Then induced cells were injected into the brain of mice,by tracking induced cells and observing the changes in the mouse brain to explore the reprogramming of fibroblasts to nerve.Method:1.Culturing fibroblast cells:Foreskin were taked from Yuhuangding Hospital,from which the foreskin to human foreskin fibroblasts were isolated;then take 2-5 passaging to transfect cells in logarithmic growth phase.Human fibroblast cell lines,mouse fibroblast cell line L929 was purchased from the Chinese Academy of Scienceslibrary of Shanghai cells.2.Lentivirus infects fibroblast cells:Fibroblast cells were seeded in 12-well plates(40000cells/well).When the cell fusion rate reach 50%,the medium was changed by 200?l fibroblasts medium + 300?l neural stem cell medium and plus virus solution and Polybrene,then plus VC and VPA at 24h,plus neural stem cell medium at 48h and after 72h,the virus-containing culture meduim was sucked out and neural stem cells meduim were injected to inducing cells.At the beginnng,the optimum condition of Lentiviral transfection expriment must be worked out.3.Inducing to nerve cells:After the viral transfection,we use puromycin to get GFP+ cells,and the medium was changed every other day.The morphological changes of cells were observed and recorded under a microscope;meanwhile we should record the cells expression of green fluorescent4 Detect nerve cells:Select good state cells,add the culture medium of neurons and glial to induce them to differentiate into neurons and glial cells.When inducing them into neurons,add RA after cultivating 2 days,add BDNF after 10 days and the medium was changes every day;when inducing them into glial cells,the medium was changed by every other day.5 Animal experiment:the induced cells were injected into the brain of mice,reared 2w,then track cells.Results:1 A large number of human foreskin fibroblast cells,human fibroblast cells,L929 cells were successfully prepared.2 After lentivirus transfecting,we use puromycin to select ST3205 infected cells(GFP+ cells),at last access all of cells expressing GFP.3 After transfection,closely observe changes in cell morphology,camera the induction cells after 7d,14d,21d,28d,35d,38d,40d,42d,and we found significant changes in the cells morphology.4 Induction cells which were cultured in neuron medium can express MAP-2,NeuN,?-tubulin.Induction cells which were cultured in glial cells medium can express MBP,GFAP.5 Expression of green fluorenscent cells were observed after paraffin sections.Conclusion:In this study,a large number of human foreskin fibroblast cells,human fibroblast cells,L929 cells were successfully prepared.And we successfully do lentivirus transfection;use virus particles which express ST3205 infect fibroblast cells to obtain miRuA+ cells,then culture cells in nerve cell medium to reprogram nerve cells.