Established Model Of Bone Marrow Mesenchymal Stem Cells Co-Cultured With Dorsal Root Ganglion Neurons

Part1SD rat Schwann cells, DRGn, SCGs, BMSCs primary cultureObjective To establish a series of easier culture systems with long-lived,highly purified, stable,to provide the guarantee for the followin g cell co culture experiments of accuracy.Methods1. Schwann cells primary culture from SD rat:The sciati c nerve and brachial plexus from1day old rats were obtained unde r stereoscopic dissecting microscope,0.125%trypsin and0.03%typ e I collagen enzyme were used for further digesting. Then F12/DM EM1:1medium (stimulating factor forskolin and bovine pituitary e xtract included) together with antimitotic agent and serum-free medi um were used to culture and purify Schwann cells. Identification an d purification of Schwann cells were evaluated by immunocytochem istry stain for S100(a marker for Swan cells) and hoechst staining.2. DRGn primary culture from SD fetal rat:dorsal root ganglion (DRG) were taken out from embryonic rats under dissecting micros copes and digested with trypsin.Then Neurobasal(NB) medium toget her with anti-mitotic drugs were used to obtain pure DRGs.Identific ation and purification were evaluated by neurofilament protein immu nocytochemistry stain and Hoechst nuclear staining respectively.3. BMSCs primary culture from SD rat:From adult SD rat femur and tibia, femur and tibia with scissors to cut in the middle, flushin g the marrow cavity was BMSCs, and cultured by adherent to the purifi ed BMSCs, cultured to the eighth generation (P8) BMSCs for the ana lysis of CD29, flow cytometry and CD44, CD45expression to identif y BMSCs and calculates its purity.Results1. Schwann cells primary culture from SD rat:Schwann cell s could survive healthily and have a good growth speed in F12/DM EM1:1medium (stimulating factor forskolin and bovine pituitary e xtract included) with a high purity of98%.2. DRGn primary culture from SD fetal rat:primary cultured DRGs are able to grow well in NB medium containing nerve growth fact or (NGF) for up to45days, and the purification rate was up to95%.3. BMSCs primary culture from SD rat:Analyzed by flow cyto metry, P8BMSCs antigen expression of high uniformity,strong positiv e expression of CD29and CD44, CD45expressing cells less, can sati sfy the need of this studyConclusion The cultivation of the primary culture from SD rat (Incl uding:Schwann cells, DRGn,BMSCs) is good activity, high purity, appl ies to the co-culture experiments. PART2Lentivirus mediated GFP green fluorescent protein labeled BMSCs and BMSCs co-culture respectively with DRGn Lentivirus mediated GFP green fluorescent protein labeled BMSCsObjective Investigate lentiviral vector mediated GFP green fluoresc ent protein labeled BMSCs suitable conditions, to obtain stable and high expression of GFP BMSCs. Lay the foundation for dynamic observation of the co-culture experiments.Methods using lentiviral vector and packaging plasmid carrying a GFP green fluorescent protein gene were transfected into293T cells, packaging containing green fluorescent protein gene GFP lentivirus, then determination of virus titer, respectively, to the value of MOI was10,20,40BMSCs infected cells, inverted fluorescence micros cope to observe the infection efficiency and fluorescence intensity o f each group and then the morphological changes, MTT method wa s used to detect the infection on the proliferation of BMSCs infecti on, so as to determine the optimum conditions.Results MOI=10transfection efficiency of (62.2±2.3)%, MOI=20transfection efficiency was (93.2±1.8)%, MOI=40transfection eff iciency not significant increase. Infection has not effect on the pro liferation of BMSCs.Conclusion MOI value of20is the suitable conditions of lentivir al vector mediated GFP markers BMSCs, GFP and green fluorescen t protein stably expressed in BMSCs, Proliferation and differentiatio n ofB MSCs was unaffected. BMSCs co-culture respectively with DRGnObjective To establish a model of BMSCs and DRGn GFP labele d co culture.Methods GFP green fluorescent protein labeled BMSCs co-culture with DRGn,then observe time and number of BMSCs differentiate i nto Schwann cell-like cell under fluorescence microscope.Results Schwann cell-like cell were observed after BMSCs co-cult ure with DRGn for3days; Typical Schwann cell-like cell were o bserved after co-culture for7days. As the culture time prolonge d, the number of Schwann cells increased.Conclusion BMSCs differentiate into Schwann cell like cells induce d by DRGn, this model can be used to study BMSCs differentiate int o Schwann cell like cells.