Studies On Human Amniotic Cells Transplantation Combined With Melatonin Administration To Treat Mouse Parkinson Disease Induced By MPTP

Parkinson disease(PD) is a common neurodegenerative disease among aging populations,and the incidence tends to increase year by year.The etiopathogenisis of PD is not clear up to now,but many factors such as oxidative stress,mitochondria disorders, apoptosis and hereditary factors,may be involved in the initiation of the disease,which caused the degeneration of dopaminergic neuron in nigrostriatum system to initiate clinical symptoms.The popular method in clinical is adapting chemical pharmacotherapy to treat PD,and most of them curative effect is not ideal and has much side effect,so it is urgent requirement to search for new therapeutic tools and methods.Discovery of neural stem cells(NSCs) is the breakthrough in the neuroscience field in last nineties,especially the finding of NSCs in adult brain disclosed a new page for the neuroscience research.In the brains of patients suffered from aging and aged-associated neurodegenerative diseases,there are damaged neurons as well as abnormal neurogenesis. It suggests that the failure of neurogenesis is associated with neurodegenerative pathological changes.So cell transplantation and drugs activating the endogenous neurogenesis in the brain have been the focus of neuroscience research.Up to now,cells used for transplantation included embryonic neural stem cells, embryonic stem cells and bone marrow stem cells.Such shortcomings as limited cell sources,carcinogenicity,ethics,etc,obstruct the progress of their clinical applications. Human amniotic cells(HACs) originated from the discard amniotic membrane of healthy pregnant women.HACs posses the advantages of lower immunogenicity,free of ethics, especially the neurobiology characteristic and functions demonstrated by recent researches, which made it a reliable cell source for regenerative medicine.In the present study,we observe the neurobiology characteristics of HACs through morphology and molecular biology methods.Although many researches have certificated that the transplanted cells can survive in the host,about 90%of them will end in death and apoptosis.The main reason is the microenvironment disorder caused by oxidative stress.Melatonin is a natural occurring compound secreted by pineal gland,with well-known antioxidant properties and indirect antioxidative function.As an endogenous neuroendocrine hormone,MT can easily pass through the biomembrane such as blood brain barrier and cytomembrane,and have high affinity to central nervous system.Additionally it has satisfied absorption rate,no obvious adverse effect and can be administrated through multiple routines,which made MT a wide application prospect in the treatment of central nervous system diseases.The protection of MT has been conformed on other cells,but the effect on NSCs and neurogenesis has not been reported yet.We designed the in vivo and in vitro experiments,to investigate the effects of MT on proliferation,differentiation and survival of NSCs.In addition,we combined it with HACs transplantation to treat MPTP-PD mice,attempting to find the possible mechanism.PartⅠ:Effects of MT on the Viability and Differentiation of Rat Midbrain Neural Stem Cells1.NSCs were isolated and cultured from the midbrain of rat embryo. Immunocytochemistry and immunofluorescence results showed that NSCs and neurospheres expressed Nestin,an antigen marker of NSCs.Also high-level telomerase activation was observed by PCR-ELISA method.So it indicated that they were neural stem cells/precursor cells.2.Compared with control,MT significantly increased both the total numbers of neurospheres and the numbers of large neurospheres under phase-contrast microscope. By MTS assay,the values of OD in NSCs were obviously increased under the condition of MT(0.05,0.1 and 1nM) in proliferation culture medium.It suggested that MT could promote the proliferation of NSCs in a dose-dependent way.3.Using MTS assay,the values of OD in NSCs were obviously decreased after adding MPP~+(1,10,50 and 100μM).Incubation NSCs with MT(0.1 and 1nM) 1h before MPP~+ addition could restore the values of OD.It suggested that MT could protect NSCs against the neurotoxicity of MPP~+.4.After adding MT(1nM) into differentiated culture medium for 6d,RT-PCR, immunofluorescence cytochemistry and western blot results showed that the expression of TH was increased and GFAP was decreased.It suggested that MT could promote the differentiation of NSCs into dopaminergic neuron and inhibited astrocytes production.5.RT-PCR results showed that the two subtypes of MT membrane receptor,MT1 and MT2,were expressed in ventral midbrain NSCs.Meanwhile,MT(1nM) increased the production of BDNF and GDNF in the cultured NSCs,which may involve in the effect of MT on NSCs proliferation and differentiation.PartⅡ:Studies on the Neurobiological Characteristics of Human Amniotic Cells1.The specific protein markers of neurocyte such as NeuN,MAP2,β-Tubullin-Ⅲ,TH CNPase and GFAP were expressed in human amniotic membrane and cultured HACs, which indicated that human amniotic membrane possessed the neurobiological characteristics.2.The pluripotent NSCs specific makers like Nestin,Musashi-1,Vimentin and PSA-NCAM,were expressed in the human amnion membrane and HACs.In addition, immunohistochemistry and immunofluorescence staining showed that cultured HACs which expressed several NSCs specific markers mentioned above,are also double positive stained with BrdU.Meanwhile,we detected Nestin mRNA in cultured HACs by RT-PCR.All the results suggested that human amniotic membrane possessed the characteristics of NSCs.3.GFAP mRNA were detected in cultured HACs by RT-PCR,and double stained for BrdU and Tubulin,TH,CNPase or GFAP positive cells presented in cultured HACs, which indicated the pluripotent potency of HACs.PartⅢ.Study on the Effect of HACs Transplantation Combined with MT Administration to Treat PD Mouse Induced by MPTP1.PD model was successfully established by injecting MPTP(i.p.,15mg/kg for 4 times, with 2h interval) on male C57BL/6 mice.Compared with control group,both the rotarod time(either at 16 rpm or 20 rpm) and spontaneous movement times of model group were significantly reduced,and escape latency and swimming distance in Morris water maze test were significantly prolonged. 2.Histomorphological observation showed both the neurons in substantia nigra and striatum zone of model mice were obviously damaged,which were manifested by severely decrease of Nissl body,condensed cellular nucleus and cytoplasm vacuolar degeneration.Immunohistochemistry and immunofluorescence results showed significantly reduction of dopaminergic neuron in the substantia nigra.3.HACs transplantation,MT administration and joint therapy can restore above behavioral and morphological changes to different degrees.4.HNA and HMA positive cells were found in the striatum of HACs transplanted and joint therapy group and some cells were PKH26/TH double positive.It indicated that some of the transplanted HACs could survive and differentiate into dopaminergic neurons.5.HACs transplantation,MT administration and joint therapy could significantly increase the production of BDNF and GDNF in the striatum,and also promote the cell proliferation of SVZ.6.The study results suggested that HACs transplantation and combination with MT exert the synergistic effect maybe by increasing the neurotrophic factors expression of striatum and promoting cell proliferation of SVZ.In summary,the present research demonstrated that MT can promote proliferation of NSCs and prevent the neurotoxicity induced by MPP~+.In addition,MT could significantly elevate TH expression,the marker of dopaminergic neurons,and decrease astrocytes maker GFAP expression,which maybe related to the increasing production of BDNF and GDNF by MT.Both HACs transplantation alone and combined with MT administration could ameliorate the symptoms of MPTP-model mice to some extent,including the disorder of motor coordination,spontaneous movement and memory.That the survival and properly differentiation of transplanted HACs,elevated BDNF and GDNF production and promotion of SVZ cell proliferation,may partly explain the therapeutic effects.This research provided the data relating to the neurobiological characteristics of HACs and MT, and explored the effects of combining with MT to treat PD,which widen the clinical therapeutic application and establish the foundation for carrying out related clinical researches.