| Neural stem/progenitor cells (Neural Stem/Progenitor Cells, NSPCs)，reside in theependymal region in central nervous system, have the ability of self-renew andmulti-differentiation into both neurons and glia. In recent years, the researches show thatthe adult mammalian brain and spinal cord continue to produce newborn neurons,whichbreak through the previous theory that neural cells loss the capacity of regeneration beforeor shortly after birth. Recently,studies point that many nerve cells, which exist in the lateralventricle wall, dentate gyrus, the SVZ, spinal cord, can regenerate. Meanwhile, neural stemcells can directionally differentiate into neurons which can form normal nervecell.Although, nerve cells will metabolize in the central nervous system, the regenerationcapacity of the nerve cells is insufficient for acute central nervous injury repair. Therefore,NSPCs transplantation has recently been focused on treating with central nervous injury.Dendritic cells (dendritic cells, DCs) is known as antigen presenting cells,whichactivate and maintain immune system. Meanwhile, DCs can secrete various cytokines toparticipate in microenvironment balance. Researches proved that DCs can promote theproliferation and differentiation of NSPCs in vitro, and enhence the activation ofendogenous NSPCs proliferation in local transplantation, thus to promote the functionalrecovery in SCI. The expression of NT-3protein and BDNF protein were detected insupernatant which collect from treatment area of DCs and NSPCs co-transplantation by ourprevious work. Huaben showed that DCs activated T cells to change immunemincroenvironment and promoted NSPCs survival.Therefore, the further study anddiscussion is necessary for interaction between DCs and NSPCs.In the present study, we postulate that interaction between DCs and NSPCspromoting NSPCs proliferation and differentiation is concerned with neurotrophic factors.We examined the expression of NT-3in co-culture system in vitro, and add specific tyrosine kinase inhibitor K252a and NT-3protein into co-culture system to observe thechanges of TrkC protein receptor of neurospheres.ObjectiveIn vitro,to study interaction between DCs and NSPCs, we focus on NT-3-TrkC signaltransduction pathway.1.To establish co-culture syestem and observe NSPCs diversify, evaluate the effect ofDCs enhance NSPCs.2. To detect the expression of Neurotrophins include NT-3,BDNF,NGF in supernatantat different time and group.3. To detect the expression of TrkC protein on the surface of NSPCs after co-culture.Materials and methods:1．DCs CultureDCs were cultured from bone marrow of femur and tibia of SPD rats (4–6weeks old)and sorted2-5×10~6cells per ml. To change the medium and cytokines at day2,day4and day6,collected the cells with a cell scraper (Sarstedt, Newton, NC) for adherent cells andcentrifuged on day7.The pellet was resuspended in fresh DC medium for experiments.2．NSPCs CultureNSPCs were cultured from the full brain of newborn SPD rats or EGFP transgenic SDrats,1×10~5cells per ml. Change the medium and cytokines at day3and day6,and harvestthe proliferation neuropheres for subsequent experiments at day7and passage bytrypsinization at70–80%confluency.3. NSPCs–DCs co-cultureIn co-cultures, DCs and the NSPCs were divided into NSPCs group, DCs+NSPCsgroup NSPCs/DCs+K252agroup, NSPCs/DCs group,NSPCs+NT-3group.To verifythe effect of DCs on NSPCs, single cell-dissociated NSPCs derived from the primaryneurospheres were sorted into6-well plates at a concentration of1×10~5cells/ml,2.5ml perwell, and1.5ml medium per well containing1×10~6DCs cells/ml in the upper chamber ofa0.4μm membrane-separated transwell system (Millipore, USA) in DCs/NSPCs+K252aand DCs/NSPCs group. NT-3100ng/ml(abcam，UK)was added in the NSPCs well andK252a1.0μmol/L in the DCs/NSPCs well the same medium only for7days.4.Immunofluorescence Suspended cells, adherent cells were fixation. The primary antibodies were applied:rabbit anti-Nestin polyclonal antibody (1:500; Abcam Cambridge, UK); mouse anti-β IIITubulin polyclonal antibody (1:500; Santa Cruz, USA);chicken anti-glial fibrillary acidicprotein (GFAP) polyclonal antibody (1:500; Abcam Cambridge, UK); rabbit anti-Olig2polyclonal antibody (1:500; Abcam Cambridge, UK); rabbit anti-TrkC polyclonal antibody(1:500; Abcam Cambridge, UK). The Suspended cells and adherent cells were incubatedwith the appropriate secondary antibodies. Nucleus were labeled with hoechst33342(Beyotime, China).5. ELISA AnalysisThe levels of BDNF, NGF and NT-3in the co-culture supernatants was detected with asensitive sandwich ELISA using the BDNF, NGF and NT-3Enzyme immunoassay System(Boster, China).According to the instructions of the manufacturer. In vitro，the expressionof NT-3,BDNF and NGF in the co-culture samples were detected, then revised according tothe total protein concentration, which was quantified by the Lowry method.6.Westrnblot AnalysisUsing Western Blot to detect the expression of TrkC protein which combine the NT-3receptor on NSPCs surface in co-culture system。7Statistical analysisThe experiment data was analysed with independent-samples single factor analysis bySPSS18.0software.Results:1. In vitro,we observed that proliferation of NSPCs in the co-culture with DCs/NSPCsgroup and NSPCs+NT-3group were more significant than DCs/NSPCs+K252a group andNSPCs group (P<0.05). The number and average diameter of NSPCs in DCs/NSPCs groupand NSPCs+NT-3group greatly increaed (P <0.05).These results suggest that DCs enhancesNSPCs proliferation in vitro.2. we found that amount of NT-3,BDNF was secreted and diliveried in supernatant ofco-culture system. The expression of NT-3reached the peak at48h. The expression of NT-3in DCs/NSPCs group and DCs+NSPCs group was more significant than DCs group andNSPCs group (p<0.05), There was no significant difference between DCs/NSPCs groupand DCs+NSPCs group was found (P>0.05). 3. The result of Immunofluorescence was that positive expression of TrkC protein onNSPCs was detected in four groups after7days.we found that expression of TrkC proteinin DCs/NSPCs group and NSPCs+NT-3group was more significant than NSPCs group andDCs/NSPCs+K252a group (P<0.05). No significant difference was between NSPCs groupand DCs/NSPCs+K252a group (P>0.05).4.Western Blot showed that the expression of TrkC on the surface of NSPCs increasedgradually in co-culture syetem. The result show that the expression of TrkC protein inDCs/NSPCs group, and NSPCs+NT-3group were siginificant increased compared withDCs/NSPCs+K252a group and NSPCs group(P<0.05). These results suggest that theexpression of TrkC was significantly different among four groups (P<0.05). This suggestedthat the TrkC protein receptor was elevated with the expression of NT-3increasely inco-culture system.Conclusion:1. In vitro, co-culture of DCs and NSPCs can increase the diameter and number ofNSPCs sphere. DCs can enhance proliferation of NSPCs.2. The expression of NT-3and BDNF can be evoked up-regulation by co-culture ofDCs and NSPCs. The secretion of NT-3reached the summit at48h. The releasing of NT-3in DCs/NSPCs group was more obviously than DCs group and NSPCs group.3. Immunofluorescence showed that the expression of TrkC protein in DCs/NSPCsgroup and NSPCs+NT-3group were significantly higher than NSPCs group and DCs/NSPCs+K252a group. This may illustrate the interactive mechanism that co-culture of DCsand NSPCs promotes NSPCs proliferation possibly by up-regulation of NT-3insupernatant of co-culture.4. Western Blot showed that expression of TrkC protein on the surface of NSPCssphere was significantly different among four groups. DCs+NSPCs group and DCs/NSPCswere more increasing than NSPCs group and DCs/NSPCs+K252a group. No significantdifference was found between DCs+NSPCs group and DCs/NSPCs group. |
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