【作者】 田有勇；

【导师】 孙圣刚；

【作者基本信息】 华中科技大学 ， 神经病学， 2007， 博士

【摘要】 第一部分VEGF对6-羟多巴胺诱导PC12细胞损伤的保护作用目的探讨血管内皮生长因子(VEGF)对6-羟基多巴胺(6-OHDA)诱导的PC12细胞变性损伤的保护作用及机制。方法用PC12细胞制作帕金森病细胞模型,采用四甲基偶氮唑盐法检测暴露于梯度浓度VEGF165后细胞的活性;流式细胞术检测对照组、损伤组(单加6-OHDA)、VEGF组(单加VEGF165)和VEGF保护组(同时加6-OHDA和VEGF165)细胞凋亡率(每组5个样本);免疫印迹法检测PC12细胞VEGF165及其受体fms样酪氨酸激酶-1(Flt-1)、胎肝激酶-1(Flk-1)、神经纤毛蛋白-1(Nrp-1)的表达,并观察VEGF165及其受体中和抗体对VEGF165作用的影响。结果随着VEGF165的浓度增加到50～100 ng/ml时,PC12细胞吸光度由0.14±0.06逐步上升至0.35±0.08(P <0.01),随后又稍下降;细胞凋亡率VEGF保护组(29.65%±6.63%)低于损伤组(57.10%±5.66%),但高于VEGF组(3.67%±1.51%)和对照组(4.76 %±1.06%),P <0.01;免疫印迹实验检测到PC12细胞有VEGF165及Flt-1和Nrp-1的表达,而未检测到Flk-1;抗VEGF165和抗Nrp-1中和抗体可阻断VEGF165的作用,而抗Flt-1抗体无作用。结论VEGF165可保护PC12细胞因6-OHDA造成的损伤, Nrp-1受体在其中发挥重要作用。第二部分腺病毒介导的VEGF165基因转移保护6-羟多巴胺诱导的PC12细胞损伤目的探讨腺病毒介导的血管内皮生长因子(VEGF)基因转移对6-羟多巴胺(6-OHDA)诱导的多巴胺能细胞损伤的保护作用。方法用腺病毒载体携带VEGF165基因(Ad-VEGF165)感染PC12细胞,并设腺病毒携带的β-半乳糖苷酶基因(Ad-LacZ)感染组和磷酸盐缓冲液(PBS)处理组作对照,基因转移成功后,用6-OHDA处理细胞,另加设不用6-OHDA处理的正常对照组。四甲基偶氮唑盐法(MTT)测定细胞活性,免疫荧光细胞化学法检测细胞酪氨酸羟化酶(TH)表达,高效液相色谱检测细胞分泌功能。结果腺病毒在30 MOI以上感染强度时能高效感染PC12细胞。Ad-VEGF165感染组的PC12细胞经6-OHDA处理后吸光度A570 (0.31±0.07)高于Ad-LacZ感染组(0.15±0.07)和PBS处理组(0.13±0.05),但低于正常对照组细胞(0.55±0.10),P < 0.01;免疫荧光染色显示Ad-VEGF组细胞胞浆平均荧光强度(86.75±21.62)高于Ad-LacZ组(51.53±17.49)及PBS组(54.19±15.82),但低于正常对照组(110.39±24.21)(P < 0.05);Ad-VEGF感染组细胞培养液中多巴胺和去甲肾上腺素含量也高于Ad-LacZ感染组和PBS处理组。结论腺病毒介导的VEGF基因转移对6-OHDA诱导的多巴胺能细胞损伤具有保护作用。第三部分腺病毒介导的VEGF基因转移对帕金森病大鼠多巴胺能神经元的保护作用目的探讨血管内皮生长因子(VEGF)基因转移对帕金森病(PD)大鼠多巴胺能神经元的保护作用。方法携带VEGF165基因的腺病毒(Ad-VEGF165)载体感染大鼠一周后,纹状体注射6-羟基多巴胺制作大鼠多巴胺能神经元损伤的PD模型。采用大鼠旋转行为观察、免疫组织化学方法和高效液相色谱技术检测VEGF165基因转移的PD大鼠行为学变化、黑质纹状体酪氨酸羟化酶(TH)、层粘蛋白(laminin)和胶质纤维原性酸性蛋白(GFAP)的表达以及纹状体区多巴胺(DA)及代谢产物含量变化。并与重组腺病毒载体(Ad-LacZ)组及磷酸盐缓冲液(PBS)组进行比较。结果Ad-VEGF165成功感染PD大鼠并高表达目的基因和蛋白。感染Ad-VEGF165的大鼠获得行为学改善,其黑质和纹状体TH阳性细胞数和纤维密度与对侧半球的比值(0.42±0.11和0.56±0.10)高于Ad-LacZ组(0.20±0.10和0.28±0.09)和PBS组(0.22±0.13和0.24±0.08),P<0.01;纹状体区血管和胶质细胞数量均明显增多;Ad-VEGF165感染组大鼠纹状体区DA及代谢产物含量与对侧比值也高于对照组。结论Ad-VEGF基因转移能保护PD大鼠多巴胺能神经元,血管和胶质细胞的增生可能参与了VEGF对在体多巴胺能神经细胞的保护机制。第四部分纹状体内VEGF基因转移挽救帕金森病大鼠多巴胺能神经元目的观察血管内皮生长因子基因转移能否挽救帕金森病(PD)大鼠多巴胺能神经元变性损伤。方法先采用6-OHDA注射制备纹状体损伤的大鼠PD模型,一周后,腺病毒载体携带VEGF165基因(Ad-VEGF165)感染PD模型大鼠纹状体,观察大鼠旋转行为及黑质和纹状体区酪氨酸羟化酶(TH)免疫组织化学变化。并与重组腺病毒载体(Ad-LacZ)组及磷酸盐缓冲液(PBS)组进行比较。结果腺病毒(Ad)感染PD大鼠后高表达目的基因和治疗蛋白;Ad-VEGF165感染组动物行为学获得改善,同时其黑质和纹状体TH阳性细胞数和纤维密度与对侧半球的比值(0.34±0.13和0.38±0.14)高于Ad-LacZ组(0.21±0.10和0.25±0.08)和PBS对照组(0.22±0.13和0.20±0.11),P<0.01。结论Ad介导的VEGF基因转移能部分挽救PD模型大鼠多巴胺能神经元,抑制其变性损伤。更多还原

【Abstract】 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.更多还原