【作者】 杨钰楠；

【导师】 高云华；

【作者基本信息】 第三军医大学 ， 影像医学与核医学， 2008， 博士

【摘要】 背景与目的Vykhodtseva首次发现超声波能开放血脑屏障(Blood brain barrier,BBB),开创了利用超声技术穿颅骨治疗的新领域,且对脑组织损伤甚微,扩大了无创性开放血脑屏障的研究前景。这一超声技术不同于以往的超声治疗领域(超声碎石、高功能超声肿瘤消融、声动力治疗等),而是利用具有诊断作用的超声波频率和强度在一定剂量的超声造影剂(又称微泡造影剂,简称“微泡”)作用下无创性开放辐照区的脑部血脑屏障,且这种作用为可逆性开放,达到了最低危险程度开放血脑屏障。故超声联合微泡开放血脑屏障成为近年国内外研究的热点。血脑屏障本身的特殊解剖学结构达到了对外来物质通过的严格选择性,从而维持了脑内环境的稳定,确保脑的正常代谢和生理功能,对人体大脑起到了很好的保护作用。在脑部疾病的治疗方面,BBB又被认为是阻止化疗药物进入脑组织进而影响疗效的主要原因,导致抗癌等药物无论是静脉注射还是动脉灌注,都难以在脑组织达到有效浓度,不能充分发挥疗效,如原发性脑瘤或继发性脑瘤等疾病的治疗,均需要药物直接在脑部发挥作用。因此药物在脑组织的疗效不仅取决于病灶对药物的敏感性,更主要的是取决于血脑屏障对药物的通透性,从有创性鞘内或脑室穿刺直接给药产生的技术操作要求较高且感染风险的限制性应用,到对药物进行脂溶性化学修饰、改变分子量大小和电荷,设计出对BBB内皮细胞的天然载体如葡萄糖、氨基酸、肽类载体,均是具有高度亲和力的水溶性药物,但是该领域的研究进展缓慢,目前还难以广泛应用于临床。于是无创性促进药物跨过BBB,使之在脑内能达到有效的浓度,是神经医学发展的重点。而通过超声波技术构建多功能的跨BBB药物转运系统也逐步走向新的超声治疗研究领域。超声联合微泡辐照脑组织,是基于静脉注射用于增强血管显影的微泡造影剂后,以一定强度与频率照射脑部,通过一系列示踪手段与检测方法证实血脑屏障开放,达到无创性可逆开放血脑屏障。目前认为,联合应用超声与微泡开放BBB主要是超声波与微泡之间相互作用的结果,其中最主要的生物学效应为空化效应,即在超声波的冲击、湍流、强辐射压作用下,微泡在负压下膨胀,并在随之而来的正压下迅速崩溃,产生内爆,此时微泡附近的温度升高,BBB开放。以上关于超声联合微气泡开放BBB的确切机制尚不明确,还需进一步的深入研究论证,但可以肯定的是,其原理不同于高渗性溶液和其他有创性开放BBB的方法。本研究的目的是通过建立体外血脑屏障模型,探讨超声联合微泡辐照作用下,血脑屏障通透性发生改变的机理与安全性,结合动物在体实验研究,对比分析超声联合微泡对BBB的影响,为将来的临床治疗提供试验依据。方法:1.小鼠脑微血管内皮细胞(Brain microvasular endothelial cell,BMVEC)的分离与传代培养:BALB/c小鼠脑组织块经0.1%胰酶消化后,筛网机械分离,最后不同密度Percoll液分层得到脑微血管内皮细胞,在含有高浓度内皮细胞生长添加剂的内皮细胞专用培养基纯化培养后传代,得到稳定的脑微血管内皮细胞源;倒置显微镜常规观察细胞形态及生长规律;BMVEC接种于涂有明胶的盖玻片上,待生长呈单层后采用链霉亲和素生物素过氧化物酶复合物法(Strept Avidin Biotin-peroxidase Complex,SABC)检测内皮细胞特有VIII因子表达,鉴定分离培养的细胞是否为内皮细胞;透射电子显微镜观察细胞内部结构;四甲基偶氮唑比色实验(Methyl thiazolyl tetrazolium,MTT)描绘细胞生长曲线。2.将BMVEC培养在具在特殊质材和孔径的Transwell小室上建立BBB体外实验模型;试漏实验初步判断BBB的形成;实时电阻测量BBB体外模型跨内皮细胞电阻;体外BBB模型HRP实时通透性实验;细胞内部结构、表面形态与分布、紧密连接蛋白-1(Zonula occludens protein 1,ZO-1)行透射电镜、扫描电镜、激光共聚焦显微镜检测。建立体外经人颞骨标本血脑屏障细胞模型:颞骨片经福尔马林消毒后涂抹上耦合剂置于细胞小室上,模拟经颅骨超声声窗条件,供体池注入微泡后进行超声辐照。3.超声联合微泡开放血脑屏障通透性的可逆性及安全性评价:应用水听器对研究中所应用的超声探头进行声学相关参数的测定,筛选出最佳超声频率与强度,为研究超声引发相关生物学效应提供量化考查指标;探讨跨内皮细胞电阻(Transendothelial electrical resistance,TEER)实时测定与通透性发生改变的关系;实验后细胞再培养3 d,观察表面形态结构改变以及电镜观察超微结构的变化,以考察微泡介导下超声辐照对血脑屏障通透性影响的发生机制与安全性;免疫组化检测紧密连接ZO-1蛋白表达是否与通透性相关;酶标仪检测辣根过氧化物酶(Horseradish peroxidase,HRP)的通透量,描绘通透曲线,探讨通透性增加的规律。4.超声联合微泡提高BBB模型通透性的机理研究:根据分组在模型中加入自制的脂膜氟烷超声造影剂(微泡),约2.0×107个左右微泡;采用经颅超声诊断仪辐照,时间10 min。分为4组,每组4个样本:(1)对照组;(2)微泡组;(3)超声组;(4)超声联合微泡组。经上述处理后,光镜和电镜观察BBB细胞膜及超微结构改变,免疫组化检测紧密连接ZO-1蛋白表达;在不同的时间点检测TEER和HRP的通透性,探讨可逆性开放BBB的机理。5.微泡介导诊断超声波辐照促顺铂跨血脑屏障转运的实验研究:根据是否加入微泡和超声辐照,分为四组:(1)对照组;(2)超声组;(3)微泡组;(4)超声联合微泡组。每组添加浓度为5ng/μl的生物素胶体金50μl和顺铂,顺铂达到9μg/cm2,供池定容在460μl,分别于超声辐照后再培养48h,并于再培养不同时间点从受池中取样,待所有取样结束后,采用高效液相色谱(High performance liquid chromatogram,HPLC)检测顺铂的通透量;透射电镜观察细胞膜与细胞内超微结构的变化以及胶体金的分布;实验前后细胞小室行扫描电镜能谱分析。6.伊文思蓝(Evans-blue, EB)示踪实验:Sprague-Dawlty (SD)大鼠,固定麻醉消毒铺巾,去除大鼠颅骨,加载经处理后的人颞骨标本;将各组动物按分组给予相应处理30 min后开胸,插管,灌注,直至从剪开的右心房内流出的液体变澄清,开颅取出脑组织,称重,浸于含甲酰胺液体中,淬取,并用DU800紫外分光光度仪测定淬取液在EB溶液最大吸收峰处的光密度值得到EB的通透量。7.超声联合微泡促顺铂跨血脑屏障转运的体内实验研究:同EB检测实验过程与步骤处理后,解剖开颅,冰台上取出脑组织,迅速在液氮中冷冻,称重后加入提取液,在冰浴上研磨匀浆,离心20 min,取上清液行HPLC进样分析。8.硝酸镧示踪实验:经颅超声联合微泡引发血脑屏障通透性改变的机理研究,配制硝酸镧固定液与漂洗液;超声辐照结束后,各实验组大鼠在超声波照射后不同时点麻醉、开胸、插管,硝酸镧灌注固定液进行灌注,灌注后快速将动物以保鲜袋包好后4℃过夜,将过夜的动物取出,组织块大小为1 mm3,随之将组织块进行相应处理包埋,半薄切片定位制成超薄切片,铀染色,铅染10s,透射电镜观察,拍照。通过体外血脑屏障模型的建议与研究,在体实验结果进行对比分析,探讨超声联合微泡对BBB的影响及促药跨膜转运作用。主要结果及结论:1.成功建立体外血脑屏障模型:BMVEC培养至汇合后具有典型的“铺路石”样外观;扫描电镜显示细胞形成致密单层,透射电镜、ZO-1蛋白免疫组化证实细胞间形成光滑、连续、高密度的紧密连接;3H葡萄糖的通透率与实时电阻呈负相关,内皮细胞电阻随着通透性的增加而减低,通透率最低时跨细胞电阻为(346±10)?·cm2。建立的BBB体外模型在形态、电阻和对大分子物质通透性方面具备了BBB的基本特性,掌握好电阻与通透性变化的动态规律,能够为各类大分子物质跨BBB能力的体外研究机制提供参考价值。2.光镜和扫描电镜显示微泡介导下超声波辐照,四组样本细胞表面无明显形态学改变和损伤,透射电镜证实超声联合微泡组细胞间紧密连接向桥粒连接过渡,超声组紧密连接减少,但没有明显细胞连接分离现象;ZO-1紧密连接蛋白免疫组化检测于超声联合微泡组局部出现减弱甚至消失,超声组无明显改变;HRP通透率检测显示超声联合微泡组BBB通透率呈波浪形递增与递减,18h后完全恢复,超声组BBB通透性增加,于30 min内恢复,通透率最高时TEER降低至(179±8)?/cm2,随着HRP通透量的减低,TEER逐渐恢复;电镜、HRP通透率以及ZO-1蛋白表达在微泡组和对照组没有明显改变。表明:微泡联合诊断超声波介导下能通过短暂开放紧密连接,降低细胞间电阻,提高通透率,达到可逆性开放血脑屏障。3. 2MHz、0.6w/cm2、10 min的诊断超声联合微泡(1μl/ml)能够促顺铂跨体外血脑屏障模型的转运,透射电镜观察到内皮细胞胞饮小体增多,细胞内可见胶体金分布;单纯超声组细胞间隙可见少量胶体金分布,对照组和微泡组细胞内外均不能见到胶体金;扫描电镜能谱分析,各组碳和锌元素总量都没有发生改变,但在超声组和超声联合微泡组两种元素的能谱分布发生转变。经HPLC检测,顺铂通透量在超声组与超声联合微泡组高于对照组与微泡组,组间比较有显著差异。表明:微泡联合诊断超声波能通过短暂开放紧密连接,促进药物顺铂跨血脑屏障的转运。4.透射电镜观察在体硝酸镧示踪实验标本:对照组及微泡组镧颗粒仅位于血管腔中,均匀附着于毛细血管内壁,血管外间隙无硝镧颗粒沉积,紧密连接未见开放,神经纤维间亦未见有镧剂沉积;超声组在电镜下可见除血管腔有镧颗粒沉积外,毛细血管的内皮下层、基底膜、神经髓鞘间可见镧颗粒沉积,神经元形态正常,胞膜完整,胞质分布均匀,细胞器正常;超声联合微泡组能见到血管紧密连接的开放,镧剂沿着开放的紧密连接处连续渗出,但外渗的镧剂并未在周围出现明显的聚集,而是沿整个血管断面在血管外均匀分布,神经细胞内未见镧颗粒沉积,神经元胞核轻度均质化,胞内细胞器结构基本正常。5.顺铂在体实验检测结果:超声联合微泡组能明显增加顺铂跨血脑屏障通透性(P<0.01),而超声组也能促进一定量的顺铂通透性,与微泡组、空白组相比没有显著性差异,这一结果与前期体外实验结果相一致。术后不同时间点取样检测顺铂通透量结果显示2 h后对顺铂通透量再次升高,并于4 h后达到最高值,与其余三组有显著性差异(P<0.01)。更多还原

【Abstract】 BackgroundVykhodtseva has been demonstrated that the ultrasound pulses can temporarily disrupt the blood brain barrier (BBB) with negligible associated effects to the brain firstly, this phenomenon which could be exploited for a non-invasive means for ultrasound technology on transcranial therapy, broaden the research at lowest risk. Ultrasound could potentially disrupt the barrier in a volume that conforms to the desired anatomical site. Ultrasonic effects totally different from its former used in the fields of (Extracorporeal shock wave lithotripsy, High intensity focused ultrasound, Sonodynamic therapy), when choose some diagnostic ultrasound combined with an ultrasound contrast agent (also equal to microbubble), could temporarily open the BBB and reversablely. This method challenges the risk limit on BBB function. Ultrasound combined with microbubbles (UCM) is well-recognized as a powerful tool, widely applied in several series of research on BBB.The blood brain barrier serves to protect the central nervous system from blood-borne infections and toxic agents. Its unique features, such as tight junctions, low vesicular transport, and high metabolic activity, accomplish the barrier function and maintain the homeostasis of the brain parenchymal microenvironment. While beneficial, the physiological characteristics of BBB make the treatment of systemic agents into brain particularly difficult. A variety of approaches have been undertaken to open the BBB for facilitating drug delivery into brain without highly invasive procedures such as opening the cranium. For example, the BBB has been opened with intra-arterial injection of hyperosmotic solutions such as mannitol. This action of mannitol causes the endothelial cells to shrink resulting in an opening of the tight junctions for lasting a few hours. However, both osmotic and chemical methods require invasive intra-arterial catheterization and produce diffuse, transient BBB opening within the entire tissue volume supplied by the arterial branch that is injected. Likewise, lipid soluble solvents (such as high-dose ethanol or DMSO), alkylating agents (like etoposide and melphalan), immune adjuvants, and cytokines, have been used to disrupt the BBB. Localized drug delivery with disruption of the BBB can be accomplished only by injecting through a needle or catheter directly into the targeted brain area. Such direct injections not only are invasive and require opening the skull but cause non-targeted penetration of brain tissue, even to carry the risk of brain damage, bleeding, and infection. There are ways to enhance propagation through the barrier: Chemical modification of the drugs or the use of other carriers such as amino acid and peptide carriers can increase transport through the BBB. This type of localized BBB disruption hardly can be accomplished clinically until further application. Previous studies have demonstrated that many ultrasound techniques can be used to increase BBB permeability. The way of ultrasound to open BBB makes it a promising tool for targeting drug delivery.A number of animal studies have demonstrated that local BBB disrupt is possible under burst ultrasound exposures and intravascular micro-bubbles. While the exact mechanisms for the disruption are not known, it is presumably related to the interaction between the ultrasound fields, the interaction between microbubbles and the ultrasound field is strongly affected by the ultrasound frequency. For example, the frequency has a large effect on the inertial cavitation threshold and by the growth of microbubbles within the ultrasound field during sonication. This could be caused by bubble collapse with associated jet formation that punctured the vessel wall.Several hypotheses on the mechanism of BBB disruption with MBs and ultrasound have been proposed. This interaction creates a change of the pressure in the capillary to evoke biochemical reactions that trigger the opening of the BBB. Therefore, we conducted the present experiment with a non-invasive way to increase BBB permeability, and to further estimate the relationship between UCM enhanced BBB permeability, also including security studies. Hence, we utilized mouse brain microvessel endothelial cells isolated and grown in monolayer culture on porous support as a BBB model in vitro, associate with animal research data to support a useful reference on clinical therapy.Methods:1. Endothelial cells were isolated from BALB/c mice brain capillaries enzyme digest, mechanical separation and density centrifugal. In brief, the meninges of mice brain were removed and the cerebra were transferred to a pre-chilled glass homogenizer. Supernatant was carefully separated from the vasculature-enriched pellet, after 2–3 washes with the mixture of homogenization buffer and 30% dextran–70 solution (3:4), resuspend the pellet very gently and plate the cells at density of one brain equivalent. BMVECs monolayers grown in collagen-coated, inner cell structure were scanned by electron microscope. VIII factor detected by the methods of Strept Avidin Biotin-peroxidase Complex (SABC); BMVECs were plated on the Lab-Tek chambered slides and grown until confluence (2–3 days); Growth line described by the depicted of Methyl thiazolyl tetrazolium (MTT).2. Brain endothelial cells are grown on cell inserts; many porous membranes for cell culture are available in different materials and pore sizes. The pore size of these membranes is 8.0μm. The permeable membranes divide the transwell device into two compartments: the inner and outer chamber. The distributions of typical tight junction proteins Zonula occludens protein 1 (ZO-1) in mouse brain microvascular endothelial cells were studied using confocal microscopy, tight junction between cells scanned by the transmission electron microscope; A constant TEER value across the cell layer will obtained and to estimate the function of BBB in vitro. The effect of the human skull on the ultrasound beam propagation was using a single-element transducer what investigated through a piece of excised human skulls and held stationary in the transwell. There’s an ultrathin acoustically and optically transparent plastic layer between the skull and transwell.3. Probe conditions will be detected by the hydrophone and choose the optimizing acoustic parameter, support the quantum data during analysing. Safety estimate will undergoing by the TEER detect and BMVECs re-culture after ultrasound exposure. Assessment of BBB permeability: was used for the HRP permeability study to investigate the paracellular diffusion across the cell monolayers.4. Mechanism research: BBB in vitro model will be devided into four groups, control group, ultrasound group, microbubble group, UCM group. The ultrasound contrast agent (self-made) contained microbubbles (approximately 2.0×107). Ultrasound waves were generated by transcranial Doppler transducer, cell ultrastructure, ZO-1 protein, TEER and HRP peameability will be detected to estimate the feasible mechanism on BBB open.5. Twenty-four transwells were divided into four groups randomly: UCM group, ultrasound group, microubbbles group and control group. Each group added 50μl colloidal gold (500ng/μl) and 9μg/cm2 Diamminedichloroplatinum (DDP). The DDP permeability was detected by the High Performance Liquid Chromatogram (HPLC) technique; The ultrastructureof brain microvascular endothelial cells (BMVEC) and colloidal gold distribution were observed under transmission electron microscope (TEM); Spectrum analysis were used to detect the two important trace elements of Carbon and Zinc on the BBB.6. Evans blue tracer experiment: the human skull covered on the rat, which original temporal bone removed. Quantitative evaluation of Evans blue dye was performed after exposure process and cardiac open, using saline to filling the heart before the atrium run off clear liquid. Each brain tissue sample was weighed, homogenized in a three-fold volume of 50% trichloroacetic acid solution, and centrifuged. The supernatants were diluted with ethanol (1:3), and fluorescence was quantified by using Ultraviolet light (UVL) reader Sample value calculations were based on Evans blue dye standards mixed with the same solvent. Results were expressed in optical density (OD) value of Evans blue dye per-milligram of tissue.7. DDP in vivo permeability: same experiment as Evans blue tracer set up described, quantitative evaluation of DDP detected by HPLC.8. Lanthanum nitrate tracing was employed to observe the mechanism which BBB permeability increased after ultrasound exposures on the presence of microbubbles. Transmission electron microscope (TEM) was used to assess the microstructure of the brain.Result:1. BBB model in vitro: the tested BALB/c mouse brain microvascular endothelial cells (BMVECs) grown on collagen-coated and fibronectin-treated culture dishes retained the morphological characteristic of microvascular endothelial cells. Typical BBB character formed a continuous monolayer with an elongated, spindle-shaped morphology, a useful tool on trans-BBB research. Electron microscopic examination, the confluent BMVECs grew into a confluent monolayer on top of the collagen fibronectin-coated transwell inserts with apparent intercellular unique W-P body.2. Tight junction transform to bridge junction. After exposure to ultrasound with the absence of microbubble, the tight junction between the endothelial cells transformed to bridge junction. Tight-junction formation decreased in the group solo-ultrasound exposure, but not apparently separation between cells. HRP permeability on group ultrasound combined with microbubble display a fluctuation wave after exposure and could recover in 18 h. TEER decreased at the lowest (179±8) ?/cm2, follows back at the same time while HRP decreased. Indicates: this technique could successfully use on transient BBB open and separate the tight junction between cells.3. Group UCM and group ultrasound sonicated by TCD, with 2MHz、0.6w/cm2 and 10 min exposure. We found that the ultrastructure of BMVEC in group UCM changed greatly from pinocytosis increased to Colloidal gold distribution in the cells. Minor Colloidal gold exist betweent the cells in group ultrasound, none in the cells, neither found in the group control and microbbles; Spectrum analysis indicates the group UCM and group ultrasound could effect the spectrum distribution of these trace elements Carbon and Zinc, but not significant difference in the group control and group microbubbles. BBB permeability of DDP increased greatly in the group UCM and group ultrasound, significantly difference among the four groups. Indicates: our results show that transcranial ultrasound associated with MBs effectively increases the permeability of DDP on the BBB in vitro, which may open the BBB reversibility.4. Lanthanum nitrate trace: In the group control and group microbbles, lanthanum only stays in the blood vessel, none in the cells and exist along the cell basal membrane, but not break through it. In group UCM, we could find the Lanthanum distribution along the BMVEC basal membrane and through the break of tight junction outside into the brain tissue.5. BBB permeability of DDP increased greatly in the group UCM, group ultrasound also increased the permeability in some extent, but only group UCM has significantly difference among the four groups (P<0.01).更多还原