The Mechanism Of BDNF Modified Human Umbilical Cord Mesenchymal Stem Cells Derived Dopaminergic Like Neurons In PD Rats

BackgroundParkinson’s disease(PD)is a common neurodegenerative disease in the elderly.Its main pathological changes are selective degeneration and necrosis of dopaminergic neurons in substantia nigra and decrease of dopamine(DA)neurotransmitter content in striatum.At present,the treatment of Parkinson’s disease is mostly symptomatic relief in order to improve the life quality of patients with PD.However,the therapeutic effect is limited.Exogenous dopaminergic neurons may provide a new and effective treatment for PD patients.Human umbilical cord mesenchymal stem cells(hUC-MSCs)are one of the typical adult stem cells.Compared with bone marrow and embryo derived stem cells,hUC-MSCs have the advantages of low immunogenicity,non-invasive collection,easy to obtain and expand in vitro.hUC-MSCs can differentiate into dopaminergic like neurons in vitro.Transplantation of dopaminergic like neurons into the brain can also improve the motor symptoms of PD rats,but most of the transplanted cells cannot survive for a long time.Brain-derived neurotrophic factor(BDNF)is a member of the neurotrophic family,which is widely distributed in various parts of the brain,and has the function of protecting nerve and promoting nerve regeneration.Studies have shown that BDNF can promote the differentiation and maturation of hUC-MSCs into dopaminergic like neurons,and improve the survival rate of differentiated neurons.However,the mechanism of BDNF modified hUC-MSCs derived dopaminergic like neurons in PD rats are still unclear.ObjectiveTo explore the possible mechanism of BDNF modified human umbilical cord mesenchymal stem cells derived dopaminergic like neurons in PD rats.Methods1.In vitro study: Mesenchymal stem cells from neonatal umbilical cord were isolated by tissue-sticking method and subcultured.The P3 generation cells were induced to differentiate into dopaminergic like neurons and transfected with p EGFPN1-GFP-BDNF plasmid or BDNF siRNA.The cells were divided into five groups: the hUC-MSCs group,the dopaminergic like neurons group,the dopaminergic like neurons + empty vector group,the dopaminergic like neurons + BDNF overexpression vector group,the dopaminergic like neurons + BDNF siRNA group.The expression of miR-1 was detected by RT-PCR.The expression of HSP60,TLR4 and My D88 were detected by Western blot(WB).2.In vivo study: PD rat model was established by injecting 6-hydroxydopamine(6-OHDA)into the right striatum of SD rats.Forty-eight PD rats were randomly divided into 6 groups(n=8 for each group): the PD group,the PD + vehicle group,the PD +hUC-MSCs group,the PD + dopaminergic neuron-empty vector group,the PD +dopaminergic neuron-BDNF overexpression vector group,the PD + dopaminergic neuron-BDNF overexpression vector + intracerebral injection of Trk B antibody group.According to the stereotactic map of Paxinos rat brain,the sites were selected for transplantation.The above groups were injected with DMEM/F12 medium,hUCMSCs,dopaminergic neuron + empty vector,dopaminergic neuron + BDNF overexpression vector,dopaminergic neuron + BDNF overexpression vector + Trk B antibody respectively.The PD group as the control group was only operated without injection.After 6 weeks of transplantation,the rats were decapitated.The substantia nigra and striatum were taken.RT-PCR was used to detect the expression of miR-1.And Western blot was used to detect the expression of HSP60,TLR4 and My D88.Results1.RT-PCR results showed that in vitro,the expression of miR-1 in the dopaminergic neuron group,the dopaminergic neuron + empty vector group,and the dopaminergic neuron + BDNF overexpression vector group was significantly higher than that in the hUC-MSCs group(P<0.01).The expression of miR-1 in the dopaminergic neuron + BDNF overexpression vector group was significantly higher than that in the other groups(P < 0.01).There was no significant difference between the dopaminergic neurons group and the dopaminergic neurons + empty vector group(P > 0.05).In vivo study,the expression of miR-1 in the substantia nigra and the striatum in the PD + dopaminergic neuron empty vector group,the PD + dopaminergic neuron-BDNF overexpression vector group and the PD + dopaminergic neuron-BDNF overexpression vector + intracerebral injection of Trk B antibody group was higher than that in the PD group and the PD + vehicle group and PD + hUC-MSCs group(P < 0.01).The expression of miR-1 in the PD + dopaminergic neuron-BDNF overexpression vector group was significantly higher than that in the other groups(P < 0.01).There was no significant difference among the PD group,the PD + vehicle group and the PD+ hUC-MSCs group(P > 0.05);2.WB results showed that in vitro,the expression of HSP60,TLR4 and My D88 in the dopaminergic neuron group,the dopaminergic neuron + empty vector group and the dopaminergic neuron + BDNF overexpression vector group was significantly higher than that in the hUC-MSCs group(P <0.01).The expression of HSP60,TLR4 and My D88 in the dopaminergic neuron + BDNF overexpression vector group was significantly higher than that in the other groups(P<0.05).There was no significant difference between the dopaminergic neurons group and the dopaminergic neurons +empty vector group(P > 0.05).In vivo study,the expression of HSP60,TLR4 and My D88 in the substantia nigra and the striatum in the PD + hUC-MSCs group,the PD + dopaminergic neuron-empty vector group and the PD + dopaminergic neuron-BDNF overexpression vector group was higher than that in the PD group and the PD + vehicle group(P < 0.05).The expression of HSP60,TLR4 and My D88 in the PD + dopaminergic neuron-BDNF overexpression vector group was significantly higher than that in the other groups(P <0.05).There was no significant difference between the PD group and the PD + vehicle group(P > 0.05).Conclusions1.In vivo and in vitro,the expression of miR-1 increases in groups with BDNF overexpression,and decreases significantly when BDNF was inhibited,suggesting that there may be a positive feedback mechanism between BDNF and miR-1.2.BDNF modified hUC-MSCs derived dopaminergic like neurons may activate Hsp60-TLR4 / My D88 signaling pathway in PD rats.