The Effect And Mechanism Of The Transplantation Of Peripheral Blood-derived Mesenchymal Stem Cells With NTRK1 Over-expression In A Rat Model Of Parkinson’s Disease

Background:Mesenchymal stem cells(MSCs)are being widely usedin clinical trial since 1995.They have three cellular properties:(1)adherence;(2)CD73+CD90+ CD105+and CD14 CD34-CD45" CD 19-HLA class Ⅱ-,(3)pluripotentfor mesenchymal lineages including osteocytes,adipocytes,and chondrocyte.Administrationof MSCs has been proposed as an innovative approach and is proved by a number of clinical trials to a certain degree for the therapy of many diseases,which are incurable by current treatment,including heart disease,neuron degenerative diseases,hematologic malignancies,autoimmune diseases,etc.The mechanism of MSCs therapy is also widely studied for regenerative therapy.Parkinson’s disease(PD)is one of the neuron degenerative diseases that could be treated by MSC therapy.PD is clinically characterized by a progressive degeneration of dopaminergic(DA)neurons.It is the second most prevalent neurodegenerative disease worldwide.PD patients develop several motor complications including rigidity,bradykinesia,and postural instability.PD rat model that exhibits DA neuron degeneration is a useful model to study PD disease and its therapy.MSCs have been injected to PD rat model to assess the effect on DA neurons.However,the efficacy of MSCs alone is not significant.It is of great interest in medical science research to promote the efficacy of MSC therapy.As MSCmediated therapeutic benefit are mainly due to their secretome,the bioactive molecules.Among which neurotrophic tyrosine receptor kinase 1(NTRK1)is found to promote cell differentiation and may play a role in specifying sensory neuron subtypes.It is also required for binding of nerve growth factor(NGF),thus NTRK1 is very important for nerve growth.Furthermore,it activates ERK1 by either SHC1-or PLC-gamma-1-dependent signaling pathway.There is report that elevated NTRK1 expression increases differentiation of neuron stem cell into cholinergic neurons under stimulation of NGF.For PD,NTRK1 could be one of the molecule candidates that can be released from transplanted MSCs to nearby neuron stem cells,thus can promote neuron stem cell differentiation into certain neuron type,such as DA neuron in substantia nigra(SN)in PD rat.In this experiment we investigated the effect of NTRK1 overexpressed peripheral blood MSCs(PBMSCs)on PD rat model.By overexpressing NTRK1 in PB-MSCs,which were then injected into PD rat model,we showed that DA neuron repair was increased in lesion site,rotational performance was also improved.Our results indicated that overexpression of NTRK1 in MSCs could be an optimized therapeutic way via MSCs for PD treatment.Objective:MSCs treatment is one of the hotspots of cell therapy for PD.Genetically modified MSCs are the emerging direction of PD therapy.This study was to investigate whether PBMSCs can repair the lesions of PD model rats and regulate the immuneion and whether NTRK1 gene-modified PB-MSCs can promote the above effects.(1)To investigate whether PBMSCs can be used as planting cells in PD model rats;(2)To explore the behavioral changes of PD model rats after transplantation of PBMSCs into PD model rats and the optimal period of behavioral improvement in PD model rats after transplantation;(3)To investigate the survival of DA in the brain of PD model rats after PBMSCs transplantation into PD model rats during the best period of behavioral improvement;(4)To investigate the expression level of pro-inflammatory factors in the brain of PD model rats after PBMSCs transplantation into PD model rats during the best period of behavioral improvement;(5)To investigate the survival of DA in the brain of PD model rats after transplantation of NTK1 gene-modified PBMSCs into PD model rats during the best period of behavioral improvement;(6)To investigate the expression of proinflammatory cytokines in the brain of PD model rats after transplantation of NTK1 gene-modified PB-MSCs into PD model rats at the best period of behavioral improvement.Methods:We used a number of male Sprague Dawley(SD)rats of similar body weight as experimental animals,and established a PD rat model by injecting a suitable amount of 6-OHDA into the substantia nigra and medial forebrain of the forebrain with a brain stereotaxic apparatus.One month later,the rotation performance of SD rats was induced by apomorphine,and the PD model was successfully established in accordance with behavioral behavioral changes.Peripheral blood of 5 healthy volunteers were isolated,purified and enriched to obtain PBMSCs.After primary culture and 5 subcultures,a higher number of PB-MSCs were obtained,and PB-MSCs with good growth status were used.The phenotypes of PB-MSCs were chemically identified by cloning antibodies CD34-FITC,CD45-PE,CD29-APC,and CD105-PE-Cy7.The shuttle plasmid pUC18-NTRK1 was constructed,transformed and amplified,and the recombinant adenovirus pAdenoX-CMV-NTRK1 was constructed and identified.Using PB-MSCs as a vector,PBMSCs were infected with pAdenoX-CMV-NTRK1 recombinant adenovirus to overexpress NTRK1.PBS,NTRK1,PB-MSCs,NTRK1-PB-MSCs were injected into the substantia nigra and striatum of PD rats by directed intracerebral injection;The number of rotations performence of each group of PD rats was evaluated by rotation test at 2 weeks,4 weeks,6 weeks,and 8 weeks after cell transplantation to obtain the best period of behavior change of PD rats after cell transplantation.Eight weeks after transplantation of PB-MSCs in PD rats,SD rats were perfused and fixed,and brain tissues were obtained and frozen sections.The content of TH was evaluated by tissue immunofluorescence technique and tissue immunohistochemistry to identify dopamine neurons in PD model of each group.Two weeks after transplantation of PB-MSCs in PD rats,SD rats were perfused and the brain tissues were obtained and the expression levels of pro-inflammatory factors(TNF-α and IL-1β)in PD models of each group were determined by commercially available ELISA kits.All results are expressed as mean±standard deviation.Data analysis was performed using SPSS 21.0 statistical software.One-way analysis of variance(ANOVA)was used to test the significance of differences between the groups.P<0.05 was considered as significant difference.Results:1.Characterization of human PB-MSCsAfter isolated from 5 healthy donors and plating for 72 h,the adhered cells exhibited a fusiform or polygonal shape as distinguished for MSCs.Cells then were stained for MSC markers and analyzed by Flow cytometry.They were identified as MSCs rather than hematopoietic stem cells based on the phenotype of MSCs showed in Fig.1:CD34-CD45-CD29-CD105-.These data indicated that we have successfully isolated MSCs from peripheral blood.2.Overexpression of NTRK1 in PB-MSCAdenovirus transfection method successfully transfected MSC with NTRK1,along with an empty vector transfected control,as shown in Fig.2a.The level of gene expression and protein expression were measured using RT-PCR and western blot,respectively.NTRK1 mRNA level was significantly higher in transfected MSCs than that in control group and vector alone group.Figure 2b showed that NTRK1 expression was much higher in transfected MSCs group.Protein levels were also quantified.Quantification showed it was almost 4 times higher than that in control group and vector alone group,as shown in Fig.2c.These results confirmed that the successful transfection of NTRK1 into MSCs also led elevated NTRK1 protein level.3.Effects of PB-MSC transplantation on behavioral changes of PD ratsMSCs with overexpression of NTRK1 were then transplanted into PD rat model to assess the effect on rotational behavior.After PD rat model was established,Cells were transplanted into SN via intranjgral injection.Rotational performance was tested in animals at different time points after transplantation:2,4,6,and 8 weeks.Among the 4 groups:vehicle(serum-free culturing medium),Ad-NTRK1,PB-MSC,and Ad-NTRK1-PB-MSC,we found that the Ad-NTRK1-PB-MSC group,in which the NTRK1 was overexpressed,showed the most improved performance,followed by the PB-MSC group as shown in Fig.3.The number of rotations of the PD rats was reduced.Ad-NTRK1-PB-MSC group showed significant decrease compared with control groups after 4 weeks.However,PB-MSC group also showed significant decrease compared with control groups after 8 weeks.The vehicle group,compared to the Ad-NTRK1 group,didn’t show much different.It is worth noting that Ad-NTRK1-PB-MSC group showed better improved performance when compared to PBMSC group after 4 weeks.These results demonstrated that PB-MSC can improve behavior of PD rat model,and NTRK1 overexpression in MSC can promote this effect.4.Effects of PB-MSC transplantation on dopaminergic neurons in PD ratsNeurodegeneration of DA neuron is characterized in PD.In PD rat model,6-OHDA injection led to severe degeneration ofDAneurons in the SN,as showed byTH immunostaining in Fig.4a.The effect of MSC transplantation on DA neurons was assessed by staining of TH in the SN and striatum at 8 weeks post transplantation.Quantification of the TH positive cells in SN showed more DA neurons were found in MSC transplanted groups,almost 3 times as that as counted in vehicle group,as showed in Fig.4b.There was not so much difference between vehicle group and Ad-NTRK1 group.However,the Ad-NTRK1-PB-MSC group showed more TH-immunoreactive cells than that found in the PBMSC group.TH-immunoreactive fibers in the striatum also showed the same trend,as showed in Fig.4c,d.These data indicated that intranigral MSC transplantation caused significant increase of THimmunoreactive cells in the SN,while NTRK1 overexpression PB-MSC exhibited the most potent effect,suggesting that NTRK1 overexpression could promote DA neurons regeneration in the SN in PD rats.5.Effects of PB-MSC transplantation on proinflammatory cytokine production in PD ratsMSCs have been noted for the ability of immunomodulatory effects in vivo.In order to see if PB-MSC transplantation will have immunomodulatory effects,we checked the pro-inflammatory cytokine TNF-a and IL-1b production in lesion area of all groups.As shown in Fig.5a,b,compared with the cytokine level in PB-MSC transplanted group,both cytokines showed no significant change in NTRK1 overexpressed-PB-MSC transplanted group.However,these MSC transplanted groups showed a slight decrease of production for both cytokines,compared with that in control groups.These results indicated that PB-MSC had effect on cytokine production in vivo,no matter if NTRK1 was overexpressed.Conclusion:1.After transplantation of PBMSCs into the brain of PD rats,the number of surviving DA increased,suggesting that it can be used as a planting cell for PD model rats.2.PBMSCs were transplanted into the brain of PD model rats.The increase of DA in the lesion and the behavioral changes of the PD model rats suggested that it had the effect of repairing DA.3.PBMSCs were transplanted into the brain of PD model rats,and the concentration of pro-inflammatory factors in the lesions decreased,suggesting that it has immunomodulatory effects.4.After NTRK1 modified PBMSCs were transplanted into the brain of PD model rats,the survival of DA in the lesions increased further,suggesting that NTRK1 can promote the paracrine effect of PB-MSCs,which can further promote the repair of DA.5.After NTRK1 modified PB-MSCs were transplanted into the brain of PD model rats,there was no further decrease in proinflammatory factors in the lesions,suggesting that NTRK1 modified PB-MSCs could not further exert immunomodulatory effects.6.PB-MSCs may be a novel strategy for PD therapy,and NTRK1 modification of PB-MSCs may promote the effects of PD.