The Therapeutic Effect Of Intracerebral Transplantation Of Neural Stem Cells Combination With Dopaminergic Neuron In 6-OHDA Rat Model Of Parkinson's Disease

OBJECTIVEParkinson disease(PD)is a chronic neurodegenerative disorder characterized by tremor,rigidity,and hypokinesia.The main pathology underlying disease symptoms in PD is a rather selective degeneration of nigrostriatal neurons leading to severe loss of dopamine(DA)in the stritum.At present,there's no therapy method which has satisfactory curative effect on PD.As the development of cell biology,the cell replacement stratage becomes more and more attractive.PD,with pathology changes with limited area,seems to be one of the diseases which can be cured by cell transplantation.Numerous researches show transplantation of DA neurons can improve the abnormal behavior of PD animals. However,the therapeutic effect of DA neuron transplantation in PD is not stabilize because of the low survival rate of transplanted DA neurons induced by local microenvironment of host.Neural stem cells(NSCs)are a subtype of tissue-specific progenitor cells that is capable of extended self-renewal and the ability to generate all major cell types of nervous tissue,such as neurons,astrocytes and oligodendrocytes. NSCs are considered as one of the most promising cell sources in cell therapy of central nervous systerm(CNS)diseases because of their unique superiorities including abundant resources,strong ability of proliferation,can integrate with host cells,without immunogenicity and oncogenicity,etc.Recent studies show that NSCs derived from different region of embryonic CNS have different potential of differerntiation.Compare with other NSCs,midbrain derived NSCs(mNSCs)have more tendancy to differentiate into DA neurons.Therefore,it is thought that mNSCs is the reasonable cell source for cell therapy in PD.However,only few mNSes can differentiate into DA neurons in vivo due to the effect of local microenvironment of host.Glia cell line derived neurotrophic factor(GDNF)is the most powerful trophic factor for DA neurons.However,its application is limited because of difficulty to cross blood-brain barrier.As the development of molecular biotechnology,gene therapy,as the most promising strategy to solve the problems in administrationg route of GDNF, become a new direction for therapy research of PD.In summary,in order to obtain reasonable therapeutic effect,the target of cell therapy for PD is not only replace local missing DA neurons,but more important,is to improve the local microenvironment of host,made which be fit for survival and growth of transtrantated cells and differentiated into DA neurons.Therefore,this study will explore the therapeuric effect of intracerebral transplantation of GDNF gene modified mNSCs combination with DA neurons in 6-hydroxydopamine (6-OHDA)rat model of PD.METHODS1.The coding sequence(CDS)of GDNF was emplified by RT-PCR from human astrocytoma cell line U251.By gene recombination technique,GDNF CDS was inserted into eukaryotic expression vector pEGFPN1 to construct recombinant plasmid pEGFPN1-GDNF.The recombinant piasmid was identified with restriction enzyme digestion and DNA sequencing.COS-7 cells were transfected with the recombinant plasmid by Fugene HD transfection regent.The expression of GDNF was analyzed by RT-PCR,western blot as well as immunocytochemistery.2.The ventral mesencephalon was dissected from embryonic day 14(El4)rat embryo. By mechanical separation method,the brain tissue was triturated into a fine single-cell suspension.The cells were cultured with the serum-free medium containing DMEM/F12(1:1),N2 supplement,EGF and bFGF.After primary neurospheres formed,cells were sub-cultured.The neural spheres of the third passage were identified with immunocytochemistery for Nestin.At the same time,the cells were inducted to differentiation,and the phenol types of differentiated cells were identified by immunocytochemistery forβ-Ⅲ-tubulin,GFAP and CNPase respectively.3.The mNSCs of the third passage were transfected with plasmid pEGFPN1-GDNF using the nucleofaction technique,48 hoUrs after transfection,the transfected cells were screened with medium containing G418,the positive clones were selected and proliferated and then labeled with SPIO mediated by FuGENE HD transfection reagent.The expression of GDNF was analyzed by RT-PCR,western blot as well as immunocytochemistry.Prussian blue stain and transmission electron microscopy was used to identify the SPIO particles in cells.To identify the differentiation ability of SPIO labled GDNF gene modified mNSCs,immunocytochemistry forβ-Ⅲ-tubulin, tyrosine hydroxylase and GFAP were performed after in vitro differentiation.4.The ventral mesencephalon was dissected from embryonic day 14 rat embryo.By trypsin digestion and mechanical separation,the brain tissue was triturated into a fine single-cell suspension.The cells were cultured with the medium containing Neurobasal,1%fetal bovine serum and 2%B27 supplement.The expression of DA neuron-specific genes was identified by RT-PCR.The purity of cultured cells was detected by TH immunocytochemistry assay.5.The rat models of PD were established by unilateral stereotaxic injection of 6-OHDA into rat ventral tegmental area(VTA)and medial forebrain bundle(MFB) region.For cell transplantation,the PD rats were randomly devided into DA neurons transplantation group(n=6),GFP gene modified mNSCs transplantation group(n=6), GDNF gene modified mNSCs transplantation group(n=6),GFP gene modified mNSCs and DA neurons cotransplantation group(n=6)as well as GDNF gene modified mNSCs and DA neurons cotransplantation group(n=6).Respective cells were stereotaxic injected into striatum(Str)of PD rats.Apomorphine(APO)induced rotational behavior test was performed to evaluate the therapeutic effect of cell transplantation.6.MRI scan was carried out to tracking the survival and migration of transplanted SPIO fabled mNSCs in vivo,and immunofluorescence histochemistry was conducted to identify the distribution and differentiation of transplantated cells.RESULTS1.The RT-PCR product of GDNF coding sequence was 650bp specific segment.By restriction enzyme digestion,the recombinant plasmid vector was digested into 650bp and 4700bp fragments.The DNA sequence of the 650bp fragment was identical with human GDNF CDS in GenBank.The results of RT-PCR,western blot and immunocytochemistery showed the GDNF was expressed successfully in COS-7 cells.2.7 days after primary culture,the cells derived from E14 rat embryonic mesencephalon form neurospheres which can be sub-cultured.A great many of neurospheres can obtained by successive passage.Immunocytochemistery showed the neurospheres were nestin positive,after differentiation the cells expressedβ-Ⅲ-tubulin,GFAP,and CNPase.3.The expression of EGFP was initially found 12 hours after transfection,increased remarkable 24 hours after transfection and reached a summit at 48 hours.One month after screened with medium containing G418,the positive clones were formed. RT-PCR,western blot and immunocytochemistery showed the GDNF was expressed correctly in cells.Prussian blue stain showed numerous blue stained particles in the cytoplasma of the labeled cells.Transmission electron microscopy showed vacuolar structures of different sizes under the cytoplasma within and outside of which there were highly density particles.The immunocytochemistery showed the labeled cells were nestin positive,after differentiation the cells expressedβ-Ⅲ-tubulin,TH and GFAP.4.The cell growth of DA neurons was most vigorous from the 7^thday to 11^thday of in vitro culture.RT-PCR showed DA neuron-specific genes including Nurr1,lmx1b and TH expressed in the cultured cells.Immunocytochemistry showed the percentage of TH positive neurons was as high as 85.7%.5.Results of APO induced rotation shows 39 of 70 rats(55.7%)were successfully established PD rats.After cell transplantation,behavior test showed cell transplantation can significantly amelioration abnormal rotational behavior induced by APO in PD rat.Compared with other groups,transplantation of GDNF gene modified mNSCs combination with DA neurons has the most striking therapeutic effect.The rotation number of rats in GDNF gene modified mNSCs and DA neurons cotransplantation group decreased nearly 80%compared with before transplantation (P＜0.01).6.MR images(T2W/FFE)showed that a dark signal appeared in the transplantation area,which gradually became enlargement as time went on.Immunoftuorescence histochemistry showed almost all cells in each group were stayed in situ,not migrating out of transplantation area.Most mNSCs kept undifferentiation state or differentiated into glia cells,only very few cells can differentiate into DA neurons. Compared with other groups,there were more mNSCs differentiated into DA neurons in GDNF gene modified mNSCs and DA neurons cotransplantation group.CONCLUSION1.The GDNF gene eukaryotic expression plasmid pEGFPN1-GDNF is constructed and GDNF gene modified mNSCs are established successfully.2.SPIO particles can effectively label mNSCs,and MRI detection of SPIO labeled cells is a promising method to analysis the cells following intracerebral transplantation.3.Compared with single transplantation of DA neurons or mNSCs,GDNF gene modified mNSCs and DA neurons co-transplantation can significantly improve abnormal behavior of PD rats.However,further research is needed to explore the mechanism of cell transplantation for PD.