Effect Of Olfactory Ensheathing Cells Transfected Of Glia Derived Neurotrophic Factor On Parkinson's Disease Rat

Parkinson's disease(PD)is a widespread neurodegenerative disorder among the old people,which seriously affects the health of individuals.Pathologically,PD experiences a progressive loss of dopaminergic neurons within the substantia nigra pars compacta and a reduction of dopamine in the striatum.So far,its etiology and etiopathogenesis is still unknown.Researchers proposed that heredity,environment, apoptosis and mitochondrial dysfunction were probably of the main mechanisms. Because of the aged tendency of population,the incidence of PD is growing year by year,threating the quality of life for the elder and forming an increasing economic burden for the society.There is no effective treatment up to date.Cell transplantation is a new therapeutic strategy for PD.However,the therapeutic effect of long time is inconsistent and some immune response often occurred in host brain. Recently,some researchers attempted to combine cells transplantation with gene transfer as a therapeutic application for PD.Olfactory ensheathing cells(OECs)are special population of glial cells sharing the properties with both Schwann cells(SCs)in peripheral nervous system(PNS) and astrocytes in central nervous system(CNS),in which they accompany and surround the olfactory sensory axons from their peripheral origin in the olfactory mucosa across the cribriform plate and into the olfactory nerve fiber layer of the olfactory bulb.On one hand,OECs can protect the regeneration of axons from the influence of inhibitors and the remeylinization for axons;on the other hand,OECs can synthesise several trophic factors,such as nerve growth factor(NGF),brain derived neurotrophic factor(BDNF),neurotrophin 4/5(NT-4,NT-5)et al.More and more people are interested in these characters of OECs and look on them as the main transplantable cells.So OECs may be the better candidates for biological implantation,but the number of regenerating axons distal from the injury site was still limited partly because of the limited expression of neurotrophic factors and the differences in the capacity of various axonal populations to regenerate through OECs implants.Glial cell line-derived neurotrophic factor(GDNF),a member of transforming growth factor(TGF-β)super-family,which was extracted and purified from B49 cells of adult rats by Lin in 1993,is a new kind of neurotrophic factor.GDNF is the most potent motor neurotrophic factor among neurotrophic factors found so far. GDNF has been reported not only to support the survival and differentiation of dopaminergic neuron,but also to protect and recover the dopaminergic system of substantia nigra and striatum in vivo.Nevertheless,there is a critical restriction on clinical application for GDNF because it can't pass the blood brain barrier(BBB).In this study,the models of PD made by 6-hydroxybutyrate(6-OHDA)in vivo were widely utilized.We adopted genetic technology and cell transplantation to treat PD,which is advanced in the world.At first,OECs were primarily cultured and purified in vitro.Then OECs-GDNF was built and transplanted into PD model rats. At last we investigated the mechanism of neurons protection of OECs-GDNF.Part 1 Culture,purification and biological characteristics study of OECsObjective:To investigate an effective method of purification for establishing primary cultures of OECs from the new born rat olfactory bulb and investigate the morphological changes and biological characteristics of the cultured OECs.Methods:OECs were harvested from olfactory bulbs and cultured,purified by the method of combining both the different rates of attachment among the various cells types and the Ara-c inhibition.The morphological changes of cultured OECs were observed at diferent stages and identified by nerve growth factor receptor p75 (NGFRp75)immunohistochemistry staining.We examined the effects of newborn rat OECs CM on PC12 cells treated by 100μM 6-OHDA.Cells were determined by MTT assay,Hoechst 33342/PI staining,Rh 123 staining and the ratio of bcl-2/bax mRNA expression by RT-PCR.Results:The cultured OECs presented two morphological types:fusiform, process-bearing multipolar and their processes weaved into net.OECs CM(25%, 50%,75%)was able to reduce the cellular damage in PC12 cells.CM could inhibit the disruption of mitochondrial transmembrane potential,upregulation of bcl-2 and downregulation of bax,compared with 6-OHDA alone,p＜0.05.Conclusions:The method of purification for OECs through combining both the different rates of attachment among the various cells types and the Ara-c inhibition is simple,inexpensive and practical.CM has a neuroprotective effect on 6-OHDA induced apoptosis of PC12 cells through up-regulation of the Bcl-2/Bax ratio and protection for mitochondrion.Part 2 Building engineered OECs-GDNF and its biological assayObjective:To construct recombination eukaryotic expression vector pIRES2-EGFP-GDNF and build the engineered OECs-GDNF.Methods:GDNF gene encoding fragment was amplified using RT-PCR and was cloned into the eukaryotic expression vector pIRES2-EGFP.After PCR amplification,SmaⅠ,SacⅠdigestion and DNA sequencing confirmation,the recombination eukaryotic expression vector pIRES2-EGFP-GDNF was constructed successfully.Then,we transfected GDNF into OECs with Lipofectamine^TM2000 positive-ion liposome,and built OECs-GDNF cells by using G418 culture medium to select positive clones.Finally,we assayed GDNF activation and quantity by ELISA and RT-PCR.So the OECs secreting high quantity of GDNF were genetic engineered cells.Results:PCR and DNA sequencing has verfied that the insert sequence was successfully cloned into the vector.ELISA assay demonstrated high GDNF protein secretion in OECs-GDNF conditioned medium.RT-PCR showed OECs-GDNF could up-regulate GDNF mRNA.Conclusions:Lipofectamine^TM2000 transfected pIRES2-EGFP-GDNF into OECs is convenient and has high transfection efficiency.Part 3 Therapeutic effect of OECs-GDNF transplanted into rat model of PDObjective:To study the therapeuti effect of OECs-GDNF transplanted into rat model of PD.Methods:After injecting 6-OHDA into the right medial forebrain bundle of rat for 2 weeks,we obtained the hemi-lateral model of PD.All rats were induced the rotational behavior after injecting apomorphine subcutaneously.The control group was set up by injecting isodose physiological saline as pseudo-operation rats.All the rats were divided into 4 groups:pseudo-operation group,PD model group, OECs-GDNF transplanted group and OECs transplanted group.The rotational behavior was evaluated by recording the numer of apomorphine-induced turns.We used tyrosine hydroxylase(TH)immunofluorescence staining to investigate the numbers of dopaminergic neurons in substantia nigra compacta(SNc)and the calibrated optic density value of TH positive fiber in striatum.Results:OECs-GDNF transplantation could ameliorate the rotational behavior of PD model rats,comparing with PD model group,p＜0.05.TH positive neurons in right SNc and TH positive fibers in right striatum of the PD model group significantly decreased,p＜0.01.After the transplantation OECs-GDNF,they could partly be restored,p＜0.05.Conclusions:OECs-GDNF transplantation had partial therapeutic effect on model rats of PD. Part 4 Effect of OECs-GDNF transplanted into PD rat on expression of Bcl-2/Bax proteinObjective:To observe the influence of OECs-GDNF transplantation on expression of Bcl-2/Bax protein in vivo.Methods:The experimental rats were divided into 4 groups according to part 3. In deeply anesthetized condition,ventral mesencephalic(VM)tissue were rapidly taken out on the ice,the Bcl-2/Bax protein expression was determined by Western blot.Results:Western blot showed protein expression of Bcl-2 in VM tissue of PD model rat decreased,p＜0.05.But it increased in OECs-GDNF transplantation group compared with PD model group at 8w.At the same time,protein expression of Bax increased in PD model rat and decreased in OECs-GDNF group.Bcl-2/Bax in OECs-GDNF group obviously increased compared with PD model group,p＜0.05.Conclusions:OECs-GDNF transplantation can up-regulate Bcl-2 protein and down-regulate Bax protein.OECs-GDNF transplantation could inhibit the depression of Bcl-2/Bax ratio to prevent the dopaminergic neurons from apoptosis.Conclusions and significance:In this study,OECs conditioned medium could afford significant neuroprotection against 6-OHDA-induced injury in PC12 cells via upregulation of bcl-2,downregulation of bax and thus attenuated the mitochondrial transmembrane potential loss,so the apoptosis was inhibited and the viability of PC12 cells was increased.OECs-GDNF transplantation had partial therapeutic effect on model rats of PD,which could up-regulate Bcl-2 protein and down-regulate Bax protein in PD model,inhibit the depression of Bcl-2/Bax ratio to prevent the dopaminergic neurons from apoptosis.Using the technology of cell culture,molecular clone,immunofluorescence staining,RT-PCR and Western blot,we research the neuroprotection on PC12 cells of OECs CM and the therapic effect on PD of OECs-GDNF transplantation.On cellular and molecular level,our study provided some experimental evidence on treating PD using OECs-GDNF.