【作者】 李照建；

【导师】 王任直；

【作者基本信息】 中国协和医科大学 ， 神经外科学， 2008， 博士

【摘要】 研究背景和目的动物实验和临床病人的研究资料均表明，急性脑缺血能诱导缺血区及其周围区血管保护性增生，新生血管能够增加缺血半暗区氧气和营养物质的供应，从而促进神经功能的恢复；然而，脑缺血后自体内源性促血管增殖因子表达量往往不足以诱导有效的代偿性微循环重建。因此，应用外源性促血管生成因子促进微血管增生治疗脑缺血的实验研究在近年来逐渐成为了研究热点之一。其中血管内皮生长因子(vascular endothelial growth factor,VEGF)是目前发现的最强的，也是在脑缺血实验中应用最多的促血管生成因子。大量研究表明，在急性脑缺血后VEGF在脑内有广泛的表达，在中枢神经系统VEGF不仅具有促进血管内皮细胞增殖和促进新生血管形成的作用，而且还具有直接的神经营养、神经保护以及抗神经细胞和神经前体细胞凋亡等多种作用，是治疗脑缺血等缺血性疾病颇具前景的细胞因子。另外，急性脑缺血的治疗有严格的时间窗，治疗因子早期干预治疗以挽救缺血半暗带受损细胞是基本治疗原则之一。然而，不少研究明确表明，VEGF在急性脑缺血的早期血管内应用可以增加血脑屏障(blood brain barrier，BBB)的破坏，增加微血管的渗出，诱发严重的脑水肿，甚至形成缺血灶内血肿。近年来，为了在脑缺血早期发挥VEGF最大限度的治疗作用，避免其作为通透性因子诱发脑水肿的副作用，很多学者相继做了不少有益的探索研究。这些研究主要集中在以下两个方面：(1)改良VEGF的用药途径：很多研究表明，在脑缺血早期通过脑室途径应用VEGF能够充分发挥其治疗保护作用，同时避免了其诱发BBB破坏和脑水肿的副作用。这些研究结果证实治疗途径对于VEGF在脑缺血的早期的成功应用可能是至关重要的，脑室途径可能因为VEGF不直接接触作用于血管内皮细胞从而减轻或者避免了诱发微血管的通透性增高；(2)VEGF和血管生成素(Angiopoietin-1，Ang1)联合应用：近年来很多研究还表明，Ang-1参与维持成熟血管的完整性，它的表达可抑制单层血管内皮细胞的通透性，防止微血管渗漏，是迄今为止发现的第一个作用于血管内皮细胞的抗血管通透性因子，这提示Ang-1在调节血管通透性方面发挥重要作用。也有很多研究也证明，Ang-1可以对抗由VEGF所引发的血管渗透性增加，并认为Ang-1和VEGF的平衡对于维持中枢神经系统BBB的完整性起关键作用，而在急性脑缺血打破了这种平衡从而诱发了BBB的破坏。研究内容、方法及结果尽管上述最新的研究成果证实了早期脑室途径应用VEGF或者VEGF和Ang-1联合应用在脑缺血的治疗中取得了较好的疗效。但是VEGF和Ang-1都是大分子蛋白质，无论通过何种用药途径恐怕很难透过BBB进入脑实质内发挥其药理作用。也有研究指出通过脑室内输注，由于BBB的限制，大分子量的治疗因子只是局限分布在脑室室管膜表层和脑脊液内，其分布体积不足脑体积的1％。相比之下，脑室途径应用病毒载体介导的VEGF或Ang-1基因转染可能是更好的治疗途径，因为病毒载体比大分子治疗因子具有更强的通过BBB和脑室壁的能力。而且我们实验室的前期工作已经证实成年大鼠侧脑室内输注携带增强型绿色荧光蛋白(enhanced green fluorescent protein，EGFP)报告基因的rAAV1型载体能够实现EGFP在大脑皮层、胼胝体和纹状体等较广泛脑区的转染和基因表达。本研究将采用脑立体定向输注的方法，将rAAV1-VEGF治疗载体或者rAAV1-VEGF和rAAV1-Angl的混合治疗载体通过侧脑室转染途径对大鼠大脑中动脉阻塞缺血再灌注模型(transient middle cerebral artery occlusion，tMCAO)进行基因治疗，以观察其在脑缺血再灌注损伤中的治疗作用和可能的机制。本研究共分两部分：(1)rAAV1-VEGF165单基因治疗大鼠脑缺血疗效及其机制的实验研究。在该研究中我们系统的检测了rAAV1-VEGF165脑室途径转染后在脑内的表达和脑微血管增殖的情况，并在缺血早期不同时问点对脑水含量、颅内压、脑水肿体积、脑梗塞体积和神经功能评分等项目进行了系统的检测。我们的结果表明在大鼠tMCAO脑缺血再灌注损伤前8周，通过脑室途径注入治疗载体rAAV-VEGF165基因治疗可以使VEGF165在脑内得到较高水平的表达，并可以促进脑内微血管密度的增加，但VEGF165的过度表达在脑缺血早期会诱发缺血半球脑水含量显著增加、脑水肿体积增大，从而引起颅内压显著性增高，进而加重了缺血后的继发性脑损伤，导致了脑梗死体积的扩展和神经功能评分的增加。上述结果可能与VEGF在脑缺血早期增加微血管和BBB的通透性密切相关。(2)rAAV1-VEGF165联合rAAV1-Ang1基因治疗大鼠脑缺血疗效及机制的实验研究。在该研究中我们系统的检测了rAAV1-VEGF165和rAAV1-Ang1经脑室途径混合转染后在脑内的表达情况，并对大鼠脑缺血再灌注损伤后的BBB的通透性、缺血周围区微血管的密度、缺血周围区脑血流量、细胞调亡及BBB功能性蛋白因子表达等项目进行了系统的检测，我们的研究结果初步表明，通过脑室途径在大鼠tMCAO脑缺血再灌注损伤前8周，利用rAAV-VEGF165联合rAAV-Ang1双基因治疗与rAAV-VEGF165单基因治疗相比，可以明显减轻脑缺血后BBB渗出，促进缺血灶周围区血管密度的显著增加及增加其脑血流量，增加脑保护蛋白c-fos的生成表达，减少了缺血性细胞的调亡，减少与脑水肿密切相关的4型水通道蛋白的表达。上述治疗机制可能与rAAV-Ang1协同VEGF促使具有occludin蛋白表达的在功能上更加成熟的新生微血管增殖有关。结论在脑缺血早期应用VEGF可以明显增加缺血脑组织BBB的渗出，诱发脑水肿，从而引起颅内压明显增高，加重了继发性脑损伤，故应该避免VEGF在脑缺血急性期的应用。脑室途径应用VEGF并不能避免其在脑内增加微血管渗出的副作用。通过脑室途径，rAAV载体介导的VEGF和Ang1双基因联合治疗脑缺血是很有前景的治疗方法，该方法一方面可以成功的实现治疗基因在脑内的广泛转染，另一方面外源性表达的VEGF和Ang1在脑缺血急性期能够协同促进有功能的微血管增生，抑制BBB渗出，协同发挥神经保护作用。更多还原

【Abstract】 IntroductionVascular endothelial growth factor （VEGF）, a potent endothelial cell mitogen associated with angiogenesis, is strongly induced after acute focal cerebral ischemia in rats. And it has been suggested as a promising pharmacological agent for stroke therapy due to its strong angiogenic and neuroprotective capacities. In addition, early intervention of therapeutic factors within the time window of therapy is considered as a main treatment principal for stroke to rescue the ischemic penumbra. However, a few of previous studies clearly indicated that acute VEGF therapy via intraluminal way during reperfusion significantly aggravated ischemic injury by increased leakage of blood-brain barrier （BBB） and intracerebral hematoma formation in experimental stroke. In contrast, several recent investigations have demonstrated that VEGF application via intraventricular approach at the early stage of stroke could exhibit neuroprotective effects on brain ischemia without increasing BBB permeability or inducing further cerebral edema. And several new reports also indicated that the VEGF-induced permeability effect in the acute stage of stroke could be counteracted by angiopioetin-1, which shows a synergic effect on inducing ischemic angiogenesis with VEGF. Fortunately, these results open the possibility that the intraventricular route may be a favourable way for VEGF to exert early protective effect on cerebral ischemic injury, since VEGF infusion by this way was believed to avoid acting directly on vascular endothelial cells to increase microvessel leakage. Or combination of Ang-1 and VEGF may provide a more adapted therapeutic strategy than the use of VEGF alone in the early therapy for stroke.However, it was also reported that large-molecule therapeutic agents with intracerebroventricular delivery only distributed to the ependymal surface of the ipsilateral ventricle and failed to significantly penetrate into cerebral parenchyma with a very small treatment volume less than one percent of the brain. Likewise, with a molecular weight as large as 45,000 Da and 72,000 Da, infused VEGF or Ang-1 protein in the cerebral ventricle may be unable to substantially transport across the BBB and reach its maximal efficiency in the treatment of cerebral ischemia either. In contrast, preischemic vector-mediated VEGF gene transfer via intraventricular way may be a preferable alternative for VEGF early treatment for ischemic stroke due to its increased ability to pass through BBB. In a previous study, it was reported that intraventricular infusion of rAAV1 vector carrying enhanced green fluorescence protein gene resulted in a multiple transduction in both choroids plexus and paraventricular regions of adult rat brain18. In the present study,we injected rAAVl-VEGF165 into the lateral ventricle of adult rats eight weeks before transient middle cerebral artery occlusion （tMCAO） for gene therapy and examine its possible effects on ischemic insults at the early stage of stroke.Materials and methodsPartⅠNinety-three adult male Wistar rats （body weight 180-200 g） were divided randomly into there equal groups to be injected with rAAV-VEGF (2.0×10¹⁰vg),rAAV-null(2.0×10¹⁰vg),or physiological saline （NS） respectively. And tMCAO models were induced after 8 weeks. During the following 48 hours, intracranial pressure （ICP）, brain water content, cerebral edema volume in MRI,cerebral infarct volume, modified neurological severity scores （NSS）, and VEGF level were determined respectively and compared statistically with that of the control groups.PartⅡ Ninety-three adult male Wistar rats （body weight 180-200 g） were divided randomly into two equal groups to be injected with rAAV-VEGF(1.0×10¹⁰vg)/rAAV-Angl(1.0×10¹⁰vg) or rAAV-VEGF(1.0×10¹⁰vg)/rAAV-null(1.0×10¹⁰vg),respectively. And tMCAO models were induced after 8 weeks. During the following 48 hours, VEGF and Ang-1 level, BBB permeablilty,microvessle density,the cerebral blood flow in peri-infarct zone, AQP4 expression, c-fos expression, and occludin expression were determined respectively and compared statistically with that of the control group.ResultsPartⅠIntraventricular application of rAAV-VEGF eight weeks before tMCAO resulted in VEGF overexpression and microvessle density elevation in multiple brain areas. At 24 hours following tMCAO, the rAAV-VEGF group, with VEGF overexpression in the rats brain, showed a significantly increase in ICP, brain water content, and cerebral edema volume compared with two control groups （P<0.05）. And the ICP significantly correlated with the brain water content in the infarct hemisphere in all three groups during 24 hours following tMCAO （r=0. 93, P<0. 05）. At 48 hours followingtMCAO,a 1.4-fold larger infarct volume and 1. 3-fold higher NSS were observed in the rAAV-VEGF group than both control groups （P<0.05）. PartⅡIntraventricular application of rAAV-VEGF/rAAV-Angl eight weeks before tMCAO resulted in VEGF and Ang-1 overexpression, and significantly decreased EB permeability and AQP4 expression following ischemia （P< 0.05）.In addition, the cerebral blood flow was also signicantly elevated with increased microvessle density in peri-infarct zone in rAAV-VEGF/ rAAV-Angl group compared to rAAV-VEGF/ rAAV-null group（P<0.05）. Moreover, we also observed that there were more positive cells expressing c-fos or occludin in the brain of rat coming from rAAV-VEGF/rAAV-Angl group than that from rAAV-VEGF/rAAV-null group（P<0.05）.ConclusionsPartⅠOur results indicate that intraventricular rAAV-VEGF pretreatment can result in deleterious intracranial hypertension and augment secondary ischemic insults at the early stage of tMCAO, and preischemic VEGF gene transfer via intraventricular approach may not be a favorable therapeutic strategy for tMCAO which should be adopted with caution or avoided in experimental stroke.PartⅡIn cerebral ischemia, the combination of Ang-1 and VEGF could be used early to promote the formation of mature neovessles and protect the injured cells without inducing side effects on BBB permeability. And the early intraventricualr injection of mixed rAAV-VEGF and rAAV-Angl may be a rational therapeutic strategy in the gene therapy for experimental stroke.更多还原