转铁蛋白靶向脂质体跨血脑屏障转导VEGF基因治疗大鼠缺血性脑卒中

【作者】 赵浩；

【导师】 王任直；

【作者基本信息】 中国协和医科大学 ， 神经外科学， 2010， 博士

【摘要】 脑卒中是世界上除缺血性心脏病之外的第二大死亡原因。脑卒中在不同国家之间的发病率有所不同,并且随着年龄的增长,脑卒中的发生率也不断增加。在脑缺血发生时,动脉供应的中心区域会产生低灌注,随后在其周围区域逐渐出现由于代谢和离子紊乱所引起的功能丧失但脑组织结构仍然保持完整的缺血半暗带。只要能够及时地恢复缺血半暗带的血流再灌注,可以大大减少脑缺血所导致的功能损伤。大量的动物实验和临床病人的研究资料均表明,急性脑缺血发生后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。血管内皮生长因子(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。在急性脑缺血发生后,VEGF在脑内呈现出上调表达,其不但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。但是由于血脑屏障(BBB)的存在,其可以阻挡大分子非脂质物质进入脑内,目前的研究还只是局限于通过病毒载体携带VEGF基因,通过颅内侧脑室或者脑实质注射的方式使外源基因在脑内有所表达。这样不仅是一种有创的治疗方式,而且由于使用病毒作为载体,很容易出现病毒基因与宿主基因整合从而导致一系列的副作用。因此探索一种非病毒的基因载体,能够通过静脉注射的方式实现外源基因跨BBB在脑内的广泛性表达,成为一个新的研究方向。目的第一部分构建一种高效的可以携带外源基因通过静脉注射的方式,透过血脑屏障进入脑内特异性表达目的蛋白的非病毒载体。第二部分比较不同剂量的脂质体(PEGylated liposomes, PLs),脑靶向脂质体(Transferrin targeted PLs, Tf-pCMV-PLs)和脑内GFAP启动子启动的特异性表达的脑靶向脂质体Tf-pGFAP-PLs经尾静脉注射后,在脑内及周围组织的表达情况。第三部分比较有Tf靶向和无Tf靶向的脂质体在包裹VEGF治疗基因后,治疗大鼠脑缺血的疗效。方法第一部分通过不同比例的配比,对脂质体合成过程中油相种类和油水比例、磷脂和胆固醇比例、旋转蒸发温度、超声温度和时间进行筛选化,并且通过试剂盒检测脂质体的包封率和Tf偶联率。将合成好的包裹LacZ基因的脑靶向脂质体(Tf-pCMV-PLs)经尾静脉注射至大鼠体内,通过组织化学染色检测LacZ基因在大鼠体内的表达情况,验证合成的Tf-pCMV-PLs是否可以转运外源基因跨过血脑屏障进入脑内表达。第二部分将PLs低剂量组(共包含80μg pCMV-LacZ质粒)、PLs高剂量组(共包含300μg pCMV-LacZ质粒)、Tf-pCMV-PLs组(共包含80μg pCMV-LacZ质粒)、Tf-pGFAP-PLs组(共包含80μg pGFAP-LacZ质粒)和空白脂质体组分别通过尾静脉注射到大鼠体内,24 h后行RT-PCR检测脑及周围组织LacZ基因mRNA的表达情况,48 h后通过β-半乳糖苷酶检测试剂盒检测脑及周围组织β-半乳糖苷酶活性,通过组织化学染色的方法检测β-半乳糖苷酶在脑及周围组织的分布情况,并且对脑及周围组织进行HE染色评估脂质体的毒性作用。第三部分制作pMCAO模型后2 d,将108只大鼠随机分为3组,每组36只分别注射Tf-VEGF-PLs, VEGF-PLs和生理盐水。Tf-LacZ-PLs同时被注射到大鼠模型体内作为VEGF成功转入脑内的提示。注射脂质体24 h后,行RT-PCR检测不同组脑内VEGF基因mRNA的情况,48h后通过Western Blot检测不同组脑内VEGF蛋白的表达情况,并分别在大鼠脑缺血后1、7、14和21 d根据mNSS量表进行神经功能评分。大鼠脑缺血后第21d,取脑组织进行TTC染色计算不同组间的脑梗死面积；注射5%FITC-葡聚糖溶液,脑组织冰冻切片后荧光显微镜下行微血管密度计数,比较不同治疗组间血管再生情况；21d时多普勒超声检测不同组间脑血流的情况,与脑缺血前脑血流量相比后,根据百分比的高低来评估不同治疗组微血管再生后,是否对脑血流的改变产生影响。结果第一部分脂质体合成的最佳比例为磷脂：胆固醇为1：1；脂药比为100：1；油相种类为二氯甲烷；油水比例为4：1；旋蒸温度为30℃；超声温度为10℃；超声时间为5min,10%的海藻糖可以增加脂质体的稳定性。测得的脂质体的包封率为87.24%,Tf偶联率为69%。将Tf-pCMV-PLs通过尾静脉注射到大鼠体内后可以观察到LacZ基因在脑内及周围器官的表达。第二部分药物注射24 h后,在大鼠脑中,PLs高剂量组、Tf-pCMV-PLs组和Tf-pGFAP-PLs组均可检测到不同水平的LacZ mRNA。但是在周围组织中,包括心肝脾肾肺,只有Tf-pGFAP-PLs组未检测到LacZ mRNA。组织化学染色结果表明Tf-pGFAP-PLs可以实现外源性LacZ基因脑内的弥漫性表达,而HE染色结果说明Tf-pGFAP-PLs对大鼠脑及周围组织无明显毒性作用。第三部分24 h后VEGF基因mRNA和48h后VEGF蛋白的表达量,Tf-VEGF-PLs组显著高于VEGF-PLs组和生理盐水对照组。随着时间的推移,在脑缺血21d后,Tf-VEGF-PLs组脑缺血大鼠的mNSS评分有显著性提高,TTC染色脑梗死面积明显小于对照组,微血管密度计数显著高于对照组,同时局部脑血流量显著高于对照组。结论第一部分通过工艺优化可以合成携带外源基因的转铁蛋白靶向脂质体(Tf-pCMV-PLs),其可以通过静脉系统跨过血脑屏障进入脑内进行表达,为实现颅内疾病的基因治疗奠定了基础。但是外源基因的启动子仍然需要进一步地改进,减少外源基因在周围器官的非特异性表达。第二部分Tf-pGFAP-PLs组可以实现外源性LacZ跨血脑屏障的脑内特异性弥漫性表达,避免了外周组织的非特异性表达,是一种较好的外源基因脑靶向的载体,为实现颅内疾病的基因治疗奠定了实验基础。第三部分Tf-VEGF-PLs可以增加脑缺血大鼠脑内VEGF的表达量,明显的改善神经功能活动,促进新生血管再生,增加局部脑血流量,减少脑缺血面积,是一种有效地治疗大鼠脑缺血的方法。更多还原

【Abstract】 Stroke ranks second after ischemic heart disease as a cause of death worldwide. The incidence of stroke varies among countries and increases exponentially with age. Initially after arterial occlusion, a central core of very low perfusion is surrounded by an area of dysfunction caused by metabolic and ionic disturbances but in which structural integrity is preserved (the ischemic penumbra). Depending on the rate of residual blood flow and the duration of ischemia, the penumbra will eventually be incorporated into the infarct if reperfusion is not achieved. Although thrombolysis and controlled hypothermia are an effective treatment in the acute stage, an effective treatment has not yet been established to enhance both neuroprotection and angiogenesis for the ischemic brain. Vascular endothelial growth factor (VEGF), an angiogenic growth factor, is considered a potentially useful therapeutic agent to attenuate ischemic brain injury. Previous studies have confirmed that VEGF may confer neuroprotection, promote neurogenesis and cerebral angiogenesis, accordingly improve histological and functional outcome from stroke through multiple mechanisms.The Blood-Brain Barrier (BBB) protects the central nervous system (CNS) from potentially harmful exogenous and endogenous molecules. Due to the poor permeability of the BBB, gene therapy drugs are difficult to cross it. In addition, most current gene vectors do not cross the BBB after an intra-venous(i.v.) administration and must be given via craniotomy or intracerebral injection, which are considered to be highly invasive and unable to deliver exogenous genes to global area of brain. Given the evident side effects of viral vectors, the goal of brain gene targeting technology pays more attention to the efficient, noninvasive and non-viral gene therapy of the brain.SubjectPartⅠ Preparing a kind of effective non-viral transduction vector, which can deliver exogenous gene into the brain by trans-blood brain barrier.PartⅡComparison the expression in brains and peripheral tissues of different dosage PLs, Tf-pCNV-PLs and Tf-pGFAP-PLs, which are injected from the tail vein.PartⅢComparison the therapeutic effects of Tf-VEGF-PLs and VEGF-PLs to the cerebral ischemic rats.Material and methodPartⅠBettering the synthetic process of liposomes by adjusting different proportion of materials, temperature of rotary evaporation and ultrasonic sound. We detected the encapsulation rate and Tf coupling rate by kit. Tf-pCMV-PLs were injected into rats via tail vein, and detected the expression of LacZ gene by histochmical staining to identify whether Tf-pCMV-PLs had delivered exogene into brain by crossing the BBB.PartⅡPLs low-dosage group (80μg pCMV-LacZ encapsuled), PLs high-dosage group (300μg pCMV-LacZ encapsuled), Tf-pCMV-PLs group (80μg pCMV-LacZ encapsuled), Tf-pGFAP-PLs group (80μg pGFAP-LacZ encapsuled) and blank liposomes were injected into rats via tail vein. Twenty-four hours after injection, RT-PCR was proceeded to detect the transcription of LacZ gene in the brain and peripheral tissues. Forty-eight hours after injection, the activity ofβ-galactosidase was detected in the brain and peripheral tissues by theβ-galactosidase detection kit. The distribution of the expression of LacZ gene was observed by histochemical staining and the toxic effect of liposomes was evaluated by HE staining. PartⅢTwo days after pMCAO,152 rats were distributed into 3 groups randomly, and Tf-VEGF-PLs, VEGF-PLs and saline were injected. Tf-LacZ-PLs was also injected into pMCAO rats as a marker to verify VEGF had been delivered into brain. Twenty-four hours after injection, RT-PCR was proceeded to detect the transcription of VEGF gene in the brain. Forty-eight hours after injection, Western blotting was proceeded to detect the expression of VEGF protein. On the day 1,7,14 and 21, neurological function of pMCAO rats was evaluated according to the scale of mNSS. On the day 21 after cerebral ischemia, brain ischemia area was calculated by the staining of TTC. MVD was counted by injection of 5% FITC-dextran under the fluorescence microscope to compare the number of angiogenesis. On the day 21 after cerebral ischemia, LCD was used to detect the blood flow of brain among groups.ResultPartⅠThe best proportion of synthesizing liposomes was following: phosphatides/cholesterol was 1:1, lipid/drug was 100:1, oil phrase was dichlormethane, oil/water was 4:1, temperature of rotary evaporation was 30℃, ultrasonic temperature was 10℃and duration was 5 min,10% trehalose was added to improve the stability of liposomes. The encapsulation of liposomes was 87.24%, Tf coupling rate was 69%. After the injection of Tf-pCMV-PLs into rats, an extensive expression of LacZ could be observed in the brain and peripheral tissues.PartⅡIn rat brains,24 h after injection, LacZ mRNA could be detected among PLs high-dosage group, Tf-pCMV-PLs group and Tf-pGFAP-PLs group. However, in peripheral tissues, including heart, liver, spleen, kidney and lung, only Tf-pGFAP-PLs could avoid the expression of LacZ gene. The result of histochemical staining demonstrated, Tf-pGFAP-PLs could achieve an extensive expression of LacZ gene in the brain, and the result of HE staining verified Tf-pGFAP-PLs did no harm to the brain and peripheral tissues.PartⅢThe results of RT-PCR and Western blotting demonstrated, VEGF mRNA and VEGF protein in Tf-VEGF-PLs group were significantly higher than that in VEGF-PLs group and saline control group. After 21 days of cerebral ischemia, mNSS in Tf-VEGF-PLs group was significantly increased and the ischemic area was significantly reduced. In Tf-VEGF-PLs group, the number of MVD was significantly higher than that in control group, and increased micro vessels also improve the local brain blood flow.ConclusionPartⅠTf-pCMV-PLs could be successfully prepared by the work of bettering producing technology and could trans-over the BBB to deliver exogene into the brain. This achievement made a strong foundation to realize the gene therapy of intracranial disease. Meanwhile the promoter of exogene was needed to be further refined in order to reduce the non-specific expression in peripheral tissues.PartⅡTf-pGFAP-PLs could realize the extensive brain-specific expression of exogene in the brain by trans-over the BBB, avoiding the non-specific expression in peripheral tissues. Tf-pGFAP-PLs was an excellent type of carrier for exogene entering into the brain.PartⅢTf-VEGF-PLs could increase the expression of VEGF in cerebral ischemic rat, significantly improve the neurological function, accelerate angiogenesis, increase local brain blood flow and reduce the area of cerebral ischemia. Tf-VEGF-PLs was a effective way to cure brain ischemia.更多还原