| Part I Vascular endothelial growth factor protects PC12 cells against 6-hydroxydopamine-induced degenerationObjective To approach the protective effect of vascular endothelial growth factor (VEGF) on PC12 cells against 6-hydroxydopamine-induced degeneration and its mechanism. Methods Neuronal PC12 cells impaired by 6-hydroxydopamine(6-OHDA)were used as the cell model of Parkinson's disease, MTT (methyl thiazolyl tetrazolium) was used to assay viability and the metabolism state of the PC12 cells exposed to gradient concentration VEGF165; Flow cytometry was used to analyse the apoptosis ratio of the PC12 cells among the groups of control, 6-OHDA, VEGF and VEGF plus 6-OHDA; Western blot was used to examine the express of VEGF165 and its receptors Flt-1, Flk-1 and Nrp-1 of neuronal PC12 cells, and the neutralizing antibodies of VEGF165, Flt-1 and Nrp-1 were used to block VEGF165 and the correspond receptors to observe their influence on the effect of VEGF165. Results The viability (represented by A570) of PC12 cells upgraded gradually to 0.35±0.08 from 0.14±0.06 with the increase of VEGF165 concentration to 50～100 ng/ml, P<0.01, and then declined slightly; The flow cytometry showed that the apoptosis ratio of VEGF plus 6-OHDA(29.65%±6.63%)was lower than that of group of 6-OHDA(57.10%±5.66%),but higher than that of control(4.76%±1.06%)and group of VEGF(3.67%± 1.51%),P < 0.01;Western blotting indicated that the neuronal PC12 cells express VEGF165, Flt-1 and Nrp-1, but Flk-1 has not been detected; the experiment of neutralizing antibody revealed that the antibodies against VEGF165 and Nrp-1 could block the effect of VEGF165 completely while the neutralizing antibodies to Flt-1 were invalid. Conclusion VEGF165 protects neuronal PC12 cells against 6-hydroxydopamine-induced degeneration, Nrp-1 receptor plays an important role in the process.Part II Adenoviral vector mediated vascular endothelial growth factor165 gene transfer protects dopaminergic cells against 6-hydroxydopamine-induced degeneration.Objective To explore the protective effect of vascular endothelial growth factor165 (VEGF165) gene transfer on dopaminergic cells against 6-hydroxydopamine-induced degeneration. Methods Adenovirus vector carrying the gene of VEGF165 (Ad-VEGF165) was used to infect PC12 cells , and groups of adenovirus vector carryingβ-galactosidae gene (Ad-LacZ) and phosphate buffered solution (PBS) were set for controls, and then the cells were exposed to 6-hydroxydopamine(6-OHDA)as the cell model of Parkinson's disease. Methyl thiazolyl tetrazolium (MTT) and immunofluorescence- cytochemistry were used to assay the viability of PC12 cells and the expression of the tyrosine hydroxylase (TH), and HPLC-ECD was performed to detect the secretion function of the PC12 cells. Results MTT showed that the absorbance A570 of Ad-VEGF165 group (0.31±0.07) was higher than that of Ad-LacZ group (0.15±0.07) and PBS group (0.13±0.05), but lower than that of normal control (0.55±0.10), P < 0.01; TH immunofluorescence showed that the mean fluorescence intensity in the Ad-VEGF group (86.75±21.62) was higher than that of Ad-LacZ group (51.53±17.49) and PBS group (54.19±15.82), but lower than that of normal control (110.39±24.21), P < 0.05; HPLC-ECD showed that the levels of dopamine and noradrenaline in the culture fluid of Ad-VEGF165 group increased than that of Ad-LacZ group and PBS group. Conclusions Gene transfer of Ad-VEGF165 has the effect of protection on dopaminergic cells against 6-hydroxydopamine-induced degeneration. Part III Adenovirus mediated vascular endothelial growth factor gene transfer protects dopaminergic neurons in Parkinson's disease: experiment with ratsObjective To evaluate the protective effect of adenovirus mediated vascular endothelial growth factor165 (VEGF165) gene transfer on dopaminergic neurons in Parkinson disease(PD). Methods Adenovirus vector coding for VEGF165 (Ad-VEGF165) was injected into the striatum of 16 SD rats, and adenovirus Ad-LacZ was injected into 25 rats and phosphate-buffered saline (PBS) was injejected into 16 rats as controls. Then, 6-hydroxydopamine (6-OHDA) was injected into establish PD model. X-gal staining was used to detected the expression of the report gene LacZ in the brain of the Ad-LacZ group 3 d, 2 w, and 6 w after injection, 3 rats in each time-point. RT-PCR was used to detect the VEGF165 mRNA exprssion in the striatum of rats of the 3 groups 2 weeks after injection, 3 rats for each group. Western blot were performed to check the protein expression of VEGF165 in the striatum of the rats of the 3 groups 2 weeks after injection, 3 rats for each group. A certain numbers of rats in each group underwent rotational behavioral anylysis 1,2, and 6 weeks after the 6-OHDA lesion. Immunohistochemistry was used to examine the number of tyrosine hydroxylase (TH) positive neuron in nigra substance, density of TH-positive fiber in striatum, and lamilin-positive vessel density, and glial fibrillary acidic protein (GFAP) positive glial cells. High performance liquid chromatography combined with electric chemical detection (HPLC-ECD) was performed to assess the contents of dopamine (DA) and its metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the striatum. Resultsβ-gal was expressed in the striatum of all Ad-LacZ transfected rats, showing the successful transfection of LacZ gene. The mRNA expression and protein expression of VEGF165 in the striatum were significantly higher in the Ad-VEGF165 group than in the other 2 groups. The apomorphine-induced rotation number in the Ad-VEGF165 group was 8.3 turns/min±8.7 turns/min 1 week after the transfection, then gradually decreased, and become 5.0 turns/min±4.4 turns/min 6 weeks after. The rotation number of the Ad-LacZ and PBS group were 14.7 turns/min±11.2 turns/min and 13.9 turns/min±8.3 turns/min respectively 1 week after the injection, then increased gradually, and became 20.2 turns/min±13.6 turns/min and 21.8 turns/min±11.8 turns/min respectively 6 weeks later,all significantly higer than those of the Ad-LacZ group (all P < 0.01). The ratios of TH-positive cells in the nigra substance in the transfected side was 0.56±0.10 in the Ad-VEGF165 group, both both significantly higher than those of the Ad-LacZ group (0.20±0.10 and 0.28±0.09) or PBS injection (0.22±0.13 and 0.24±0.08), (all P < 0.01). The ratios of lamilin-positive vessel desity of the transfected side to that of the contralateral side in the Ad-VEGF165 group was 2.09±0.42, and the ratio of GFAP-positive glial cells of the striatum of the transfected side to that of the contralateral side was 2.77±1.21 in the Ad-VEGF165 group, both significantly higher than those in the Ad-LacZ group (1.01±0.16 and 1.64±0.28) and the PBS group (1.04±0.09 and 1.56±0.62) (P < 0.01 and 0.05). HPLC-ECD showed that the contents of DA, HAV and DOPAC of the striatum at the lesioned side in the Ad-VEGF165 group were all significantly higher than those in the other 2 groups (all P < 0.01). The ratio to the DA, DOPAC, and HVA of the lesioned side striatum to those of the contralateral side in the Ad-VEGF165 group was 0.35±0.11, 0.46±0.09, and 0.38±0.09 respectively, all significantly higher than those in the Ad-LacZ group (0.17±0.15, 0.21±0.07, and 0.16±0.05) and PBS (0.19±0.06, 0.20±0.09, and 0.14±0.03) (all P < 0.01). Conclusion Gene transfer of Ad-VEGF165 has a protective effect on rat dopaminergic neurons of PD. The proliferations of vessels and glial cells induced by VEGF may involve in the process of neuroprotection to the dopaminergic neurons of PD.Part IV Intrastriatal gene transfer of vascular endothelial growth factor rescues the dopaminergic neurons from degeneration in a rat model of Parkinson diseaseObjective To approach the ability of intrastriatal gene transfer of vascular endothelial growth factor165 mediated by adenoviral vector to rescue dopaminergic neurons in a rat model of Parkinson's disease (PD). Methods we constructed recombinant replication-deficent adenoviral vectors carrying the gene of VEGF165 (Ad-VEGF), and injected Ad-VEGF (or Ad-LacZ and PBS as controls) into the striatum of rats at 7 days after the lesion by 6-hydroxydopamine. The rat rotational behavior analysis and tyrosine hydroxylase (TH) immunohistochemistry were performed to assess the change of dopaminergic neurons. Results The rats receiving Ad-VEGF injection displayed a significant improvement in apomorphine-induced rotational behavior and a significant preservation of TH-positive neurons and fibers compared with control animals. The ratios of TH-positive cells in the substantia nigro and the density of fibers in the striatum to the contralateral side of the rats infected by Ad-VEGF165 (0.34±0.13 and 0.38±0.14) were higher than those of infected by Ad-LacZ (0.21±0.10 and 0.25±0.18) and PBS injection (0.22±0.13 and 0.20±0.11), P < 0.01. Conclusions Intrastriatal gene transfer of VEGF165 mediated by adenovirus is able to partly rescue dopaminergic neurons and inhibit the neurodegeneration in PD models. |
PDF Full Text Request