| Parkinson's Disease(PD)is a progressively neurodegenerative disorder in middle-aged and elderly people,It is implicitly characterized by a stepwise loss of nigral dopaminergic neuron.The capacity of dopamin substantia nigra to striatum is decreased and symptoms are marked by bradykinesia,rigidity,resting tremor and postural instability.Some studies suggested that many mechanisms contributed in PD,including genetics,environment,oxidative stress,neuroinflammation,apoptosis,and so on.Currently,there is no effective cure method.Therapeutic approaches available are mainly palliative strategies and maintenance therapy required.As the disease proceeds,the pharmacotherapeutic efficacy is inevitably worn off,and implicated by side effects.Therefore,cell replacement therapy(CRT)is a promising restorative therapy intending to secure a long-lasting relief of patients' symptoms.Here,we study the differentiation of hUC-MSCs into neurons in vitro.Histological examination after hUC-MSCs transplantation to observe the bio-distribution and explore the mechanism and effect of hUC-MSCs transplantation for further clinical trials.Part I Isolated,culture,passage,and identification of hUC-MSCsObjective:To study the characteristics of hUC-MSCs and differentiation of hUC_MSCs into neurons in vitro.Methods:To isolate,culture and differentiate hUC-MSCs from umbilical cord blood.The hUC-MSCs were cultured with epidermal growth factor,basic fibroblast growth factor(GDNF),BDNF,and all-trans retinoic acid(RA)for differentiation.The characteristics is observed with phase contrast optics.Surface markers were confirmed by flow cytometry and biological markers characteristic of neural stem cells were labeled by immunocytochemical staining.Results:After passage,the hUC-MSCs obtained from human umbilical cord blood displayed distinctive vortex shape.hUC-MSCs were positive for CD44,CD90,CD29 and CD 105;negative or weak expression of CD34,CD45,and Human Leukocyte Antigen-antigen D Related,The results were confirmed by flow cytometry.hUC-MSCs nuclei were positively labeled for Nestin,NSE,and GFAP,while TH was negatively labeled by immunocytochemical staining.hUC-MSCs could be differentiate into neural cells.Conclusions:It is suggested that hUC-MSCs already had biological markers characteristic of neural stem cells.Part ? The establishment of rat PD modelObjective:To establish the successful rat PD modelMethods:The rats were fixed on the stereotaxic instrument,The coordinates for medial forebrain bundle lesions and for right ventral tegmental area.Rotations were made by intraperitoneal injections of apomorphine.Brain sections were processed for TH immunohistochemical detection.Tyrosine hydroxylase-positive cells in the SN were counted.Results:Compared the loss of the TH positive cells in SN,the implanted group was 90%higher than that in the normal group.Conclusions:Unilateral complete injured PD rat model was successfully Established by 6-hydroxydopamine injection.Part ? Experimental study of hUC-MSCs transplantation in the treatment of advanced PD ratsObjective:To investigate the therapeutic effect of hUC-MSCs transplantation on PD rats.Methods:Rats were randomly assigned to 2 groups:25 rats were assigned to the group implanted(in the striatum)with differentiated hUC-MSCs,and 21 rats were placed in the saline control group.Behavioral changes observed in the PD models.One week after transplantation,brain slices were observed with a Zeiss fluorescence microscope and processed for TH immunohistochemical detection.Tyrosine hydroxylase-positive cells were counted.From 2 to 8 weeks after cell transplantation,TH-positive cells were counted in the bilateral SN of lesioned rats.Eight weeks after transplantation,rats were chosen for analysis of DA levels in the striatum by enzymelinked immunosorbent assay.Results:One week after transplantation,about 80%± 34%of BrdU and TH-positive cells were seen in the striatum of the differentiated hUC-MSC group.From 2 to 8 weeks after cell transplantation,TH-positive cells were counted in the bilateral SN of lesioned rats.Tyrosine hydroxylase-positive cells were reduced in the SN on the lesioned side.Two months after transplantation,DA content in the striatum on the lesioned side of the brain was up to 1754.12114.79 pg/g in the transplantation group;however,the saline control group is 1687.94±14.82 pg/g.Dopamine levels were significantly greater in the transplantation groups than in the saline control group.Conclusions:The rotation scores of PD rats in the transplantation group were reduced after hUC-MSCs transplantation,and the hUC-MSCs nuclei were positively labeled for TH. |
PDF Full Text Request