| Objective:1. Establish a simple and effective rat model of Parkinson'disease by direct injection of 6-hydroxydopamine(6-OHDA) in the middle forebrain bundle(MFB) with stereotactic technique, and offer an experimental basement for the next research.2. Establish a method to isolate and culture the rat bone mesenchymal stem cells(rBMSCs).3. Study the transfect efficiency of three kinds of recombinant adenovirus including Ad-GFP, Ad-GFP-NGF and Ad-GFP-Noggin. The influence of transfection and the expression of NGF and Noggin were observed.4. The effects of chemical supplement, NGF and/or Noggin in inducing rBMSCs into neuron-like cells were discussed.5. To explore the effects and mechanisms on recovery of neurological function observed in rats of PD model grafted by rBMSCs modified with NGF and/or Noggin.Methods:1. Establish a rat model of PD by direct injection of 6-OHDA in MFB. To check if the establishment of PD rat model was successive by hypodermical injection of apomorphine.2. rBMSCs were isolated from rat bone marrow and purified through adherence ability. The cells were identified as rBMSCs by their phenotypical properties and their ability of differentiating into osteocyte and adipocyte.3. The proper MOI was chosen through recombinant adenovirus different density. rBMSCs were divided into 5 groups:rBMSCs, rBMSCs transfected with Ad-GFP, rBMSCs transfected with Ad-GFP-NGF, rBMSCs tarnsfected with Ad-GFP-Noggin and rBMSCs transfected with Ad-GFP-NGF combined with Ad-GFP-Noggin. Detect the influence of transfection to rBMSCs and the expression of NGF and Noggin through immunofluorescence.4. Induce rBMSCs differentiate into neuron-like cell and observe their expression of neuron specific marking proteins NF(H).5. rBMSCs modified by different genes were injected into the lesion side of striatum of PD model rats by stereotactic apparatus. Rotation test evoked by apomorphine was used to study the recovery of neurological deficits of rats. The distribution of survival transplanted cells was observed, and their diffentiation into neurons and astrocytes was detected through immunofluorescence. Four weeks after implantation, the number of TH positive cells in nigra was detected through immunohistochemical assessment. The contents of DA(dopamine),DOPAC(dihydroxyphenylacetic) and HVA(homovanillic acid) in striatum of model rats were detected by high performance liquid chromatography-electric-chemical discharge(HPLC-ECD).Results:1. The PD model established by injecting 6-OHDA into rat's MFB is a stable PD model. The establishment of the model had offered a basement for further experiment.2. The cells pure by their adherence ability had basic phenotypical properties of rBMSCs, and could differentiate into osteocyte and adipocyte.3. When MOI=50, Ad-GFP Ad-GFP-NGF and Ad-GFP-Noggin could transfect rBMSCs with high efficiency and low toxicity. rBMSCs transfected with recombinant adenovirus had green fluorescence autonomically at the same time it could express corresponding protein and send out red fluorescence through immunofluorescence. rBMSCs transfected with both Ad-GFP-NGF and Ad-GFP-Noggin had positive reaction to both NGF or Noggin at the same time.4. Neuron-like appearance could be seen after rBMSCs had been cultured in chemical supplement for 5h. NF(H),but astrocytic marking protein GFAP, was positive in them. Most of cells died in this supplement. The shape of rBMSCs transfected with Ad-GFP was not change. In the three groups transfected with Ad-NGF,Ad-Noggin and the two virus, the cells got the shape of neuron 72h later, some of them were positive to NF(H) and negative to GFAP. These cells grew in good state. The ratio of NF(H)+cells in combinating transfected group was the highest.5. One week after injection of the modified rBMSCs into lesion side striatum of rat model, the neurological function of rats was improved, the apomorphine-induced rotation was reduced significantly, the decrement of the combined group was remarkable. Observed by microscope, survival rBMSCs could be seen in striatum, subcortex, hippocampus which around the injective position and even the opposite side hemisphere. Two weeks after implantation, some of the survival cells were positive to NF(H) and some others were positive to GFAP. The rats transplanted with rBMSCs modified with two genes had more survival cells and higher ratio of NF(H)+ or GFAP+ cells than other groups. Four weeks after implantation, the survival rate of TH positive cells in nigra was found an increased tendency in the combined group. But the difference of each group was not statistically significant. The contents of DA, DOPAC and HVA in lesion side and uninjured side were detected by HPLC, their ratio of each group was compared. The ratio of groups that implanted with rBMSCs was higher than the group with PBS and group with no implantation. The ratio of group that implanted with rBMSCs which modified with two genes was the highest in all groups.Conclusion:1. The PD rat model established by intracal injection of 6-OHDA has good stability and repeatability.2. rBMSCs could be isolated and purified from rat bone marrow through their adherence ability and subculture. This method was simple, and would not influence the activity of cells. rBMSCs could be identified effectively by phenotypical properties and multipotency.3. Ad-GFP, Ad-GFP-NGF and Ad-GFP-Noggin could transfect rBMSCs safely. The transfected cells could express GFP, NGF and Noggin effectively.4. Chemical supplement, NGF and Noggin could induce rBMSCs into neuron-like cells in vitro. But chemical supplement interrupted the survival of cells, while NGF and Noggin could improve their differentiation.5. rBMSCs modified with NGF and Noggin could be implanted into the lesion side striatum of rat model. The transplanted rBMSCs could survive and migrate in the brain of model rats. Some of them could differentiate into cells with phenotype of neuron or astrocyte. The transplanted rBMSCs could improve the recovery of neurological function and the delivery of DA in rat model by several effective ways.
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