CAJ | 학술논문

目的探讨血管内皮生长因子(VEGF)基因治疗对大鼠脑缺血的治疗作用及其机制.方法成年雄性SD大鼠64只,分为对照组和治疗组,每组均32只,采用立体定向微量注射的方法将相同滴度的rAAVl-VEGF(治疗组)和rAAVl-Lacz(对照组)注入大鼠侧脑室,21 d后制作大脑中动脉区缺血再灌注(MCAO)模型,于不同时间点对各组动物进行神经损伤程度评分(NSS),以免疫定量分析法榆测各组大鼠脑组织中VEGF的表达量、以免疫组织化学染色检测脑内VEGF的表达部位和微血管密度(MVD),并行vWF和BrdU的免疫荧光双重标记,采用尾静脉注射FITC-葡聚糖法行脑微血管灌注成像,并对各组缺血半暗带区域的脑微血管灌注情况进行评估.结果 (1)治疗组的NSS评分有明显改善;(2)VEGF可在脑内多个结构表达,其VEGF表达量是对照组的27倍;(3)治疗组MVD为157±13、对照组为89±9(P<0.05),且治疗组半暗带区域可见大量BrdU阳性的内皮细胞,而对照组未见;(4)缺血半暗带区域微血管显影面积为(152 617±13 076)μm2/mm2,对照组为(91 658±6577)μm2/mm2(P<0.05).结论 rAAVI-VEGF可介导VEGF在大脑内表达,并对脑缺血大鼠神经功能的恢复具有促进作用,其治疗机制可能与VEGF促进新生血管形成并改善脑组织供血有关.
목적 탐토혈관내피생장인자(VEGF)기인치료대대서뇌결혈적치료작용급기궤제.방법 성년웅성SD대서64지,분위대조조화치료조,매조균32지,채용입체정향미량주사적방법장상동적도적rAAVl-VEGF(치료조)화rAAVl-Lacz(대조조)주입대서측뇌실,21 d후제작대뇌중동맥구결혈재관주(MCAO)모형,우불동시간점대각조동물진행신경손상정도평분(NSS),이면역정량분석법유측각조대서뇌조직중VEGF적표체량、이면역조직화학염색검측뇌내VEGF적표체부위화미혈관밀도(MVD),병행vWF화BrdU적면역형광쌍중표기,채용미정맥주사FITC-포취당법행뇌미혈관관주성상,병대각조결혈반암대구역적뇌미혈관관주정황진행평고.결과 (1)치료조적NSS평분유명현개선;(2)VEGF가재뇌내다개결구표체,기VEGF표체량시대조조적27배;(3)치료조MVD위157±13、대조조위89±9(P<0.05),차치료조반암대구역가견대량BrdU양성적내피세포,이대조조미견;(4)결혈반암대구역미혈관현영면적위(152 617±13 076)μm2/mm2,대조조위(91 658±6577)μm2/mm2(P<0.05).결론 rAAVI-VEGF가개도VEGF재대뇌내표체,병대뇌결혈대서신경공능적회복구유촉진작용,기치료궤제가능여VEGF촉진신생혈관형성병개선뇌조직공혈유관.
Objective To investigate the therapeutic effect of vascular endothelial growth factor (VEGF) gene expression mediated by recombinant AAV1 (rAAV1) vector in brain ischemia and the mechanism thereof.Methods Sixty-four SD rats were randomly divided into 2 equal groups and received intra-ventricular injection with rAAV1-VEGF or rAAV1-lacZ as controls.21 days later the rats underwent transient middle cerebral artery occlusion (MCAO).Neurological severity score (NSS) was recorded 1,2,3,7,14,and 21 days after MCAO.48 rats were sacrificed 21 days after MCAO and brains were taken out from 48 rats.Immune quantitative analysis was used to identify the quantity of VEGF expression,lmmunohistochemistry was used to identify the site of VEGF expression.Immunofluorescence double labeling of von Willebrand factor (vWF) and 5-bromodcoxy-uridine (BrdU) was performed to detect the proliferation of endothelial ceils.Fluoreseein isothiocyanate ( FITC ) -dextran was infused into the caudal vein of 8 rats from each group and then the rats were killed with their brains taken out to evaluate the cerebral microvessel perfusion and microvessel density.Results The NSSs of the VEGF group 7,14,and 21 days after MCAO were all significantly lower than those of the control group ( all P <0.05 ),and the VEGF165 protein expression quantity was 27 times as that of the control group (P < 0.05 ).Immunohistochemistry demonstrated that VEGF expression was distributed mainly in the caudate putamen,corpus callosum,choroid plexus,and hippocampas in the VEGF group,while no expression was detected in the control group.The microvessel density of the VEGF group was 157 ± 13,significantly higher than that of the control group [ ( 89 ± 9),P < 0.05 ].BrdU +/vWF + endothelial cells were detected in the area adjacent to the MCAO.The density of microvessel infused with FITC-dextran was (152 617 ±13 076) μm2/mm2 in the VEGF group,significantly higher than that of the control group [(91 658 ± 6577)μm2/mm2,P < 0.05].Conclusion rAAV1 mediates the VEGF gene expression in multiple structures in the brain and attenuates the neurological deficit of MCAO.VEGF gene transfer may stimulate angiogenesis and improves blood supply in brain.Neovascularization may be a therapeutic strategy for brain ischemia.