Transplantation Of Mesenchymal Stem Cells Labeled With Enhanced Green Fluorescent Protein Into The Parkinson's Disease Rat

Parkinson disease(PD) is a neurodegenerative disease due to the attition of thedopaminergic neurons of the substantia nigra and locus ceruleus. Progressive loss of theseneurons results in resting tremor, rigidity and hypokinesia. Now the incidence of PD risesstably with extension of human life.Though more and more anti-parkinson drugs emerge,the drugs only can improve symptoms, but not cure the disease and delay the progression ofPD. Moreover, long-term prescribe of levodopa can cause side effects and wearing-off. Soit is significant to investigate the etiological treatment of PD. For the loss of thedopaminergic neurons maily confined to the substantia nigra which causes the motorsymptoms, the transplantation of stem cells is very promising. Now the seed cells of thetransplantation include fetal nigral cells, embryonic stem cells, neural stem cells andmesenchymal stem cells(MSCs). MSCs has several advantages such as powerfulself-renewal, multiple differentiation ability, convenience of obtaining, no ethical issues andpromising auto-transplantation. In this experiment we firstly attempt to establish the stablemethods of culturing and expanding MSCs, then explore the its ability of differentiationinto neural cells. Finally, we will transplant the labeled stem cells into PD rats, observe theeffect of transplantation and investigate the probable mechanisms. Partâ… Isolation, identification of rat mesenchymal stem cells and itsdifferentiation into dopaminergic neuron in VitroObjective: To explore the isolation,eapansion and differentiation of rat bonemarrow-derived mesenchymal stem cells into dopaminergic neuron.Methods: Bone marrow was collected from 4-6 week-old SD rat,suspended in thecomplete culture medium of LG-DEEM/10%FCS/10%EGF/100U/mlP/S and planted intoFN-coated plates, rMSCs were purified and expanded in vitro. Phenotype and morphologyof rMSCs were analyed by FACS. Then they were assigned into 3 groups. All MSCs werecultured in the same medium in the first 5 days, and the medium in group A,B,C wasreplaced with induction medium which contained 1/2,1/3 and no striatal extract in volumerespectly in the next 10 days. The differentiated cells were detected by Nestin ,GFAP, NSEand TH immunocytochemical in 5th, 10th and 15th day. Finally the TH positive cells werecounted.Result: rMSCs are adherent ceils with morphology of fibroblastoid presentingspindle-shaped and polygonal-shaped and morphyology of rMSCs is still similar in cultureof 15 passages. Phenotype showed that rMSCs are positive for the surface markersCD₄₄,CD₉₀,CD₁₀₅ and negative for CD₄₅. Most of rMSCs become bipolar and tripolar afterderivation. Immunofluorescence showed that the induced cells were stained withNestin ,GFAP, NSE and TH. The rates of TH-positive cells were (11.9±0.5)%, (8.3±0.3)%and (1.6±0.2)%in group A,B,C. There were statistical difference between group A andgroup C.Conclusion: These results indicated that the culture rMSCs could survive and proliferate invitro and have the potentials of multi-differentiation and transversal differentiation. Striatalextract could induced rMSCs into DN. Therefore, they have a promising prospect intherapy of cell transplantation in PD. Partâ…¡Recombinant adeno-associated virus 2-mediated greenfluorescent protein expression in rat bone marrowmesenchymal stem cellsObjective: To observe the transfection rate and expression time of enhanced greenfluorescent protein(EGFP) in mesenchymal stem cells (MSCs) with recombinant adenoassociatedvirus 2(rAAV2), and the change of cell expansion after transfection.Methods: MSCs were cultured in vitro and the surface antigen expression of MSCs wasdetected, then they were assigned into 2 groups. MSCs in group rAAV₂ were mixed with0.1ml rAAV₂-EGFP at multiplicity of infection 10⁶ and 0.1ml PBS were added in groupPBS as contrast. Then there are 5 passages in culture. The expression rates of EGFP inMSCs P5 and P10 in both groups were detected by flow cytometry and the curve of cellexpansion were presented.Result: The expression rates of EGFP in MSCs P5 and P10 in group rAAV₂ were(21.71±1.02) % and (20.34±1.13)%,however, the rate in group PBS were (0.89±0.05)% and(0.96±0.07)%. There were statistical difference between 2 groups and no differencebetween same group accordingly. The curve of cell expansion discovered that MSCs ingroup rAAV₂ expanded slowlyer than in group PBS.Conclusion: The transfection rate and the expression time of EGFP in MSCs is effectiveenough to use this technique to label stem cells in vivo. Then survival, migration anddifferentiation of the transplanted cells can be observed and monitored. Partâ…¢Migration and differentiation of mesenchymal stem cells labeledwith enhanced green fluorescent proteinin the Parkinson's DiseaseObjective: To study curative effects, survival, migration and differentiation ofmesenchymal stem cells(MSCs) transplantated into the Parkinson's Disease(PD) rats.Methods: The 6-hydroxydopamine(6-OHDA) induced hemiparkinson rats were selectedand allocated randomly into 2 groups. Mesenchymal stem cells labeled by enhanced greenfluorescent protein(EGFP) and PBS were injected into the striatums of the PD ratsrespectively. The rotation tests were taken every week after transplantation.Immunochistochemical staining of Neun, GFAP and TH were carried out in the first weekand the ninth week after transplantation. The exprsssion of TH and BDNF were detected byWestern blot in the ninth week.Result: The rotation per minute(RPM) in the MSCs group and PBS group were 8.24±0.56turns/min and 8.27±0.61 turns/min respectively before transplantation, and there were nosignificant difference between them ( P＞0.05 ) However, the RPM were7.46±0.53urns/min,8.13±0.61urns/min respectively in the third week. So there wassignificant difference between two groups from the third week to the ninth week aftertransplantation (P＜0.05). Mesenchymal stem cells were noted alonge the injection tractmostly in the first week while the cells migrated to the cerebral hemispheres in the ninthweek. But most of MSCs were stilled in the injected hemisphere and some can reach thetermination of corpus callosum. No labeled cells was observed in the cerebellum and brainstem. Furthermore dysembryoma was not found in brain. Immunochistochemical stainingof Neun,GFAP were detected in the first week and in the ninth week after transplantation,but expression of GFAP was predominate. And in the ninth week expression of TH in the grafted striatum was observed by Western blot and immunochistochemical analysis only inthe MSCs group. The result demonstrates that mesenchymal stem cells can differentiateinto neuron,gliocyte and DN, but the neural differentiation potential of MSCs results inrevalent differentiation towards the astrocytic lineage. None was detected in the PBS group.In the end, there were also significant difference between two groups in the comparison ofexpression of BDNF in the grafted striatum observed by Western blot.Conclusion: It is probably safe and effective to transplantate MSCs into striatum of PDrats to correct behavioral of Parkinsonian rats by its differentiation into DN.Conclusion1. Mesenchymal stem cells not only have vigorous proliferative ability, but also candifferentiate into glial cells, neurons and the dopaminergic neurons specifically in vitro.2. EGFP- rAAV₂ can transfect mesenchymal stem cells effectively. Moreover, EGFPcan be expressived for enough time to label the stem cells and have no impact on itsproliferative ability.3. Mesenchymal stem cells can improve rotation test remarkably after itstransplantation into PD rat models, which may be induced by its differentiation into thedopaminergic neurons and nervous protective contribution.