| BackgroundInflammatory demyelination is the primary morphological hallmark characterizing muliple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis. It’s a neurodegenerative disease caused by autoimmune responses in central nervous system (CNS), influencing the health of people more than 2.5 million around the world. LINGO-1, a key negative regulator of axonal formation, inhibits axonal growth and remyelination, promotes the apotosis of oligodendrocytes and neurons. On the basis of anti-inflammation therapy, the transplantation of neural stem cells (NSCs) is regard as a promising approach for therapy MS.ObjectiveIn the current study, we acquired primary NSCs from fetal mice hippocampus through mechanical separation. NSCs were modified by transducing LINGO-1-Fc. We examined whether LINGO-1-Fc delivered by LINGO-1-Fc-NSCs can effectively attenuate EAE.MethodsMice were randomly divided into three groups:Model group (Vehicle), GFP-NSCs group, LINGO-1-Fc-NSCs. To understand the therapeutic effects of LINGO-1-Fc on EAE, LINGO-1-Fc-NSCs group were treated with 1×106 LINGO-1-Fc-NSCs in 200ul PBS by intravenous injection on days 23 post-immunization, GFP-NSCs group were treated with 1×10 GFP-NSCs, model group was given the same volume of PBS. Clinical scores were evaluated and recorded daily. All mice were sacrificed on days 48 post-immunization. Spinal cord tissues embedded in paraffin were sectioned and stained with hematoxylin and eosin(H&E)to reveal inflammatory cells infiltration, stained with Luxol fast blue to reveal demyelination. Frozen sections of lumbar spinal cord enlargement from animals were stained with immunofluorescence staining. The antibody of MBP, NeuN and GFAP were double-stained respectively with anti-GFP to reveal endogenous and exogenous nerve repair. Iba-1 was stained to illustrate the activation of microglials/macrophages, Gr-1 was stained to explain the infiltration of neutrophils. Ex vivo and in vitro, the subpopulations of CD4+T cells in CNS and spleen were analyzed by flow cytometry. In vtro,the influences of LINGO-1-Fc on the differentiation and proliferation OPCs were researched through immunofluorescence staining with 04, CNPase and Ki67.Main results are listed as follows:1. NSCs were infected with lentivirus particles (pCDH-LINGO-1-Fc-copGFP) for 48h, the concentration of LINGO-1-Fc cecreted by LINGO-1-Fc-NSCs (195.00±9.75 ng/106 cells) was significantly higher than NSCs or GFP-NSCs.2. All animals started at entering the onset on days 13 post-immunization, and entered the peak period of EAE on days 19 post-immunization.13-25days after NSCs were grafted, compared with model group, GFP-NSCs (P<0.01) reduced clinical score, but the effect time of LINGO-1-Fc-NSCs group was earlier than GFP-NSCs and its effect was obvious than GFP-NSCs. From 25 to 48 days post-immunization, compared with model group or GFP-NSCs group, LINGO-1-Fc-NSCs group dramartically reduced clinical score and significantly shorten the peak period of EAE to 6 days.3. HE staining suggested that, compared with model group or GFP-NSCs group, LINGO-1-Fc-NSCs group reduced inflammatory cell infiltration, but between GFP-NSCs group and model group had no significant difference. LFB staining showed that demyelination area in LINGO-1-Fc-NSCs group was smaller than other groups.4. The results of immunofluorescence staining are listed as followed:(1) LINGO-1-Fc-NSCs group had a higher mean fluorescence intensity (MFI) of MBP than other groups, both GFP-NSCs group and LINGO-1-Fc-NSCs group showed exogenous oligodendrocytes (GFP+/MBP+cells) differentiation, but there was no significantly differences between two groups.(2) Compared with GFP-NSCs group, LINGO-1-Fc-NSCs group promoted exogenous neurons (GFP+/NeuN+cells) differentiation.(3) Compared with model group, GFP-NSCs group and LINGO-1-Fc-NSCs group remarkably reduced the numbers of total astrocytes and endogenous astrocytes in lesion, and LINGO-1-Fc-NSCs group was less than GFP-NSCs group in terms of the numbers of total astrocytes and exogenous astrocytes.(4) Compared with model group, LINGO-1-Fc-NSCs group significantly reduced the number of Iba+cells (P<0.01), but there was no significant differences between LINGO-1-Fc-NSCs group and GFP-NSCsgroup (P=0.066).(5) Compared with model group, other two groups dramartically reduced the number of neutrophils from spinial cord.5. The results by flow cytometry analysis reflected that LINGO-1-Fc-NSCs group decreased the level of CD4+/IL-17+A cells (P<0.05) compared with model group.6. In vitro, results suggested that the supernatant of LINGO-1-Fc-NSCs inhibited OPCs proliferation and promoted OPCs differentiation to oligodendrocytes compared with the supernatant of GFP-NSCs.Conclusion1. The transplantation of LINGO-1-Fc-NSCs and GFP-NSCs can attenuate EAE, the effect time of LINGO-1-Fc-NSCs is earlier than GFP-NSCs and its treatment effect is obvious than GFP-NSCs.2. LINGO-1-Fc-NSCs have a powerful neural repair property, LINGO-1-Fc-NSCs promote endogenous oligodendrocytes maturation, exogenous neurons differentiation, inhibit OPCs and endogenous astrocytes proliferation, restrain exogenous astrocytes differentiation.3. LINGO-1-Fc-NSCs are capable of important immunomodulatory function, it can inhibit inflammatory cells infiltration, Th17 differentiation and the activation of microglias/macrophages.
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