【作者】 张震；

【导师】 薛一雪；

【作者基本信息】 中国医科大学 ， 生理学， 2009， 博士

【摘要】 前言血脑屏障（blood-brain barrier,BBB）是维持脑内物质转运平衡的重要结构,严格限制大分子物质与98%的小分子物质进入中枢神经系统。与BBB结构类似,脑胶质瘤细胞与微血管之间存在着血肿瘤屏障（blood-tumor barrier,BTB）,是位于内皮细胞之间由一些复杂的紧密连接（tight junction,TJ）蛋白系统构成的限制亲水性物质跨过的细胞旁屏障。TJ蛋白系统是由claudins、occludin和ZO-1等多种TJ相关蛋白构成的复杂连接。BTB的通透性略高于正常的BBB,但仍具有屏障的功能。BTB的存在是限制抗肿瘤药物疗效的关键。研究者们已经尝试采取多种方法提高药物通过BTB的通透性,如使用高渗性药物甘露醇、设计能跨过屏障的药物或载体、直接将药物注射入脑、用导管经动脉分支给药、经鼻腔给药等,但是这些方法因受使用剂量大、有一定创伤性、安全性低、开放时间短等诸多条件的限制而难以得到临床应用。低频超声（low-frequency ultrasound,LFU）是临床诊断中的常规手段之一。由于LFU具有在组织中易穿透,声能吸收少,对组织损伤小的特点,近年来,国内外学者也开始将LFU用于实验性治疗的研究中。LFU已经应用于骨关节病及感染和炎症的治疗、促进骨折愈合、溶栓、软组织修复及抗肿瘤细胞治疗等实验性治疗研究。最近,国外研究发现,LFU作用于脑组织可以引起BBB的开放,采用的超声频率范围为260 kHz～1.63 MHz,也有选用最大频率为3.5 MHz的报道。研究显示,LFU也可以提高药物在脑内的通透性,但目前仍处于实验阶段,超声条件还没有统一的参数标准。我们的前期研究工作表明,在1 MHz、12 mW、声压0.48 MPa和作用时间为20 s的条件下进行LFU辐照能够可逆性开放BBB的同时,对正常神经元没有产生明显损伤。考虑到肿瘤术后骨窗,便于超声辐照,上述研究提示LFU有可能成为研究可逆性开放BTB的新方法。目前关于LFU开放BBB或BTB的机制尚不清楚。有报道灌注小剂量缓激肽（bradykinin,BK）可能通过与胶质瘤细胞上的B2受体结合,选择性开放BTB,增加抗肿瘤药物卡铂到达脑肿瘤组织,有效延长脑恶性胶质瘤的生存期。但是,研究发现,BK还有一些缺点,如剂量较大时有明显的降血压作用,并且由于BK可被快速降解,使得其增加BTB通透性的时间很短暂,因而临床应用受限。因此,探寻提高缓激肽作用效果和时间的方法是目前的研究方向。本研究尝试单独应用LFU辐照,或者LFU和小剂量BK联合应用的方法,研究对BTB通透性的影响,以及开放BTB细胞旁途径的机制;同时研究二者单独应用和联合应用时,三种BTB的TJ相关蛋白claudin-5、occludin和ZO-1的mRNA和蛋白表达的变化,探讨其可能的机制,并力图寻求安全、较长效地开放BTB的二者联合应用的剂量。方法1、建立有超声透声窗的大鼠BTB模型。2、应用伊文氏兰（Evans blue,EB）渗透法检测LFU与BK单独或联合应用前后脑胶质瘤大鼠BTB渗透性的变化。3、应用透射电镜技术观察LFU与BK单独或联合应用前后脑胶质瘤微血管内皮细胞间TJ及脑胶质瘤等超微结构的改变。4、应用RT-PCR法检测LFU与BK单独或联合应用前后脑胶质瘤大鼠TJ相关蛋白claudin-5、occludin和ZO-1的mRNA表达的变化。5、应用免疫组织化学法检测LFU与BK单独或联合应用前后脑胶质瘤大鼠TJ相关蛋白claudin-5、occludin和ZO-1的分布和表达的变化。6、应用免疫荧光法检测LFU与BK单独或联合应用前后脑胶质瘤大鼠TJ相关蛋白claudin-5、occludin和ZO-1的分布和表达的变化。7、应用western blot法检测LFU与BK单独或联合应用前后脑胶质瘤大鼠TJ相关蛋白claudin-5、occludin和ZO-1蛋白表达的变化。结果1、LFU辐照后,C6脑胶质瘤大鼠的BTB通透性增加,在1.5 h达到峰值,12 h恢复正常水平。2、LFU辐照引起C6脑胶质瘤大鼠TJ开放;对神经元无明显损伤,同时诱导C6胶质瘤细胞凋亡。3、LFU辐照后,TJ相关蛋白claudin-5、occludin和ZO-1的mRNA表达显著减少;其减少的趋势为在1.5 h时达最低值,12 h以后逐渐恢复正常。4、LFU辐照后,C6脑胶质瘤大鼠脑组织中微血管内皮细胞上的TJ相关蛋白claudin-5、occludin和ZO-1的表达减少;其减少的趋势为在1.5 h时达最低值,12 h以后逐渐恢复正常。5、LFU与BK单独应用后,BTB的通透性显著增加;二者联合应用后,BTB通透性进一步增加。6、LFU与BK单独与联合应用后,透射电镜均可显示C6脑胶质瘤大鼠TJ开放。7、LFU与BK单独应用后,TJ相关蛋白claudin-5、occludin和ZO-1的mRNA表达显著减少;二者联合应用后TJ相关蛋白claudin-5、occludin和ZO-1的mRNA表达进一步显著减少。8、LFU与BK单独应用后,TJ相关蛋白claudin-5、occludin和ZO-1的蛋白表达显著减少;二者联合应用后TJ相关蛋白claudin-5、occludin和ZO-1的蛋白表达进一步显著减少。讨论本研究证实,LFU辐照能显著增加BTB的通透性,可逆性开放BTB。此过程中LFU对神经元无明显损伤;能够诱导C6胶质瘤细胞凋亡。LFU和BK单独应用和联合应用显著增加了C6胶质瘤大鼠BTB的通透性,透射电镜显示BTB上的TJ开放。同时,TJ相关蛋白claudin-5、occludin和ZO-1的mRNA和蛋白的表达水平显著下降。联合应用组中的5/6LFU+2/3BK组和LFU+BK两组与LFU组和BK组相比,BTB的通透性增加,TJ相关蛋白claudin-5、occludin和ZO-1的表达进一步下降。提示5/6LFU+2/3BK组和LFU+BK组能够更显著地开放BTB。TJ相关蛋白claudin-5、occludin和ZO-1的mRNA和蛋白的表达水平的显著下降可能是LFU和BK单独应用和联合应用开放BTB的主要分子机制之一。在前期研究中,我们证明了采用LFU能够可逆性开放BBB（power=12 mW,pressure=0.48 MPa,frequency=1 MHz,exposure deep=5mm,exposure time=20s）。有报道LFU能够提高脑组织对抗体或其它正常情况下难以入脑的药物的通透性,可以靶向性地将阿霉素、gadolinium等转运入脑。这些研究提示LFU能够可逆性开放BBB和BTB。为了进一步研究LFU增加BTB通透性的机制,本研究用透射电镜观察了与我们前期报道相同的辐照条件下1.5 h时,BTB上血管内皮细胞之间的TJ的变化,发现TJ明显开放,提示细胞旁途径—TJ的开放是BTB通透性增加的机制之一。与我们研究结果相似的有报道采用690 kHz,1 Hz,20 s参数的超声辐照,超声辐照声压在0.4 MPa～3.1 MPa范围内变化时,电镜下观察到辣根过氧化物酶（HRP）能够经跨细胞和细胞旁途径穿过血管壁。关于LFU开放BBB和BTB的确切机制,目前尚不清楚。有报道低强度超声（f=1.029 MHz,power=0.2 W）可通过血管收缩改变脑血流来改变BBB的通透性。辐照时间为20 s时起主要作用的是空化效应,造影剂引起的BBB开放可以在体内稳态空化、宽频发射及溢出都不存在的情况下发生,可能来源于超声与微泡之间的相互作用,与LFU的造影剂产生的二次及三次谐波信号的增加有关。造影剂Optison能够与LFU联合作用开放BBB,但是对脑组织几乎没有损伤。应用260 kHz-1.63 MHz LFU可逆性开放BBB或BTB时,有脑组织缺血,毛细血管红细胞漏出、神经元凋亡等副作用。本研究显示小剂量BK能显著增加BTB对EB的通透性,可选择性开放BTB内皮细胞的TJ,通过细胞旁途径选择性增加BTB的通透性。这与一些报道结果一致。BK还通过其它机制如开放K_ATP通道和K_Ca通道引起BTB通透性的增加。但是BK引起BTB通透性的增加通常是短暂的,最佳作用峰值时间为15-30 min,60 min后恢复至正常水平。有研究显示BK引起的BTB通透性的短暂增加可能是由于B2受体的神经支配导致的快速耐受所致。为了减少LFU和小剂量BK开放BBB或BTB的副作用,探讨可能的应用途径,我们在本研究中探讨了LFU和小剂量BK联合应用的可能性和相关机制。研究结果显示,在LFU和BK联合应用组,BTB通透性显著高于对照组,血管内皮细胞间的TJ开放,提示在增加BTB通透性上,存在协同作用。为了初步研究LFU和BK联合应用的可能机制,我们研究了BBB细胞旁途径的最主要结构TJ的变化。TJ是一个维持动态平衡的稳定结构,能被各种细胞外的刺激诱导快速解聚和重组。本研究应用RT-PCR和免疫荧光、组化以及western blot等方法从mRNA和蛋白表达水平证明了LFU和BK联合应用时的协同作用。关于LFU对脑血管内皮细胞间TJ的影响及机制报道不多,尚无定论。有报道LFU能通过瞬态空化效应产生的机械剪切力损伤细胞膜,引起细胞膜上的TJ相关蛋白结构的完整性破坏,蛋白表达水平减少。国外学者在LFU（1.5 MHz）辐照后4 h内,应用免疫电镜技术观察到TJ相关蛋白claudin-5、occludin和ZO-1表达减少,6-24h以后逐渐恢复正常,这与我们的研究结果相似。但是LFU引起claudin-5、occludin和ZO-1的mRNA表达水平下降尚未见报道。总之,本研究证明了LFU能增加BBB和BTB的通透性,可逆性开放BBB和BTB的TJ。引起TJ相关蛋白claudin-5、occludin和ZO-1的mRNA和蛋白的表达水平显著下降。本研究首次证明了LFU和BK联合应用在增加大鼠BTB的通透性上存在协同作用,BTB上的TJ开放。TJ相关蛋白claudin-5、occludin和ZO-1的mRNA和蛋白的表达水平显著下降。关于LFU和BK联合应用引起TJ相关蛋白claudin-5、occludin和ZO-1 mRNA和蛋白水平降低的机制有待进一步研究。本研究结果为可逆性开放BTB提供了新途径。结论1、超声参数为频率1 MHz、强度12 mW、声压0.48 MPa和作用时间20s的LFU是可逆性地开放BTB的适宜条件。2、LFU在显著地增加BTB的通透性的同时,能可逆性地开放BTB的TJ;LFU在开放BTB的条件下对神经元无明显损伤,能够诱导C6胶质瘤细胞凋亡。3、LFU辐照能使TJ相关蛋白claudin-5、occludin和ZO-1在mRNA和蛋白水平的表达显著减少,此作用与LFU诱导TJ开放相关。4、LFU与BK联合应用能以协同作用的方式显著增加BTB的通透性。5、LFU与BK联合应用能以协同作用的方式显著减少TJ相关蛋白claudin-5、occludin和ZO-1在mRNA和蛋白水平的表达。更多还原

【Abstract】 ObjectiveBlood-tumor barrier（BTB）,similar to blood-brain barrier（BBB）,is located between glioma cells and macrovessels formed by highly specialized endothelial cells. It restricts the paracellular diffusion of hydrophilic molecules to tumor tissue by an elaborate network of complex endothelial tight junction（TJ）,which contains several ubiquitous molecular components including claudins,occludin,ZO-1,ZO-2,and ZO-3. The permeability of BTB is slightly higher than that of BBB,but it is still a significant barrier for therapeutic drugs to penetrate.Various approaches have been applied to increase the permeability of BTB in chemotherapy.However,these approaches are restricted from wide clinical use because of the high dosage,invasiveness,low reliability,or short opening time of the BTB.Low-frequency ultrasound（LFU） is one of the routine diagnostic methods.LFU could penetrate through the tissue easily,is lower to be absorbed of power,and causes less damage to the tissue,so it has been used in experimental therapy in recent years. LFU has been applied to osteoarthropathy,infection,inflammation,fracture, thrombolysis,parenchyma repairment,and anti-tumor therapy.Recently a few studies have shown that LFU can open brain BBB,with frequency from 260 kHz to 1.63 MHz, and the highest frequency is 3.5 MHz.LFU increases the penetration of durgs after opening BBB,but the standard parameters of ultrasound criteria have not been obtained. In our previous study,BBB permeability increased after LFU sonication（frequency 1 MHz,power 12 mW） without detectable neuron damage.Therefore,LFU may be used to open BTB in chemotherapy.The mechanism of the effect of LFU on BTB permeability is still unclear.Low-dose bradykinin（BK） transiently increases the permeability of BTB, selectively opens BTB,possibly through its interaction with BK type 2（B2） receptor on glioma cells,and increases the transportation of carboplatin into brain tumors, without the obvious vasodilatory effect caused by high-dose BK.Low-dose BK infusion may prolong the survival time in rats with gliomas.However,the clinical use of low-dose BK is also limited because of a fast enzymatic degradation.The purpose of this study was to determine whether LFU and low-dose BK increase the permeability of BTB in a synergistic manner and whether TJ is involved in BTB permeability in C6 glioma rats.We examined the effects of LFU on BTB permeability,and determined the expressions of TJ-related proteins,claudin-5,occludin, and ZO-1.Methods1.C6 brain glioma rats model were established.2.Before and after LFU sonication and BK infusion individually or in combination,the BTB permeability was determined by Evans blue（EB） assay in vivo in rats with C6 brain glioma.3.Before and after LFU sonication and BK infusion individually or in combination,the changes in TJs and ultrastructure were observed under transmission electron microscope in BTB model in vivo in rats with C6 brain glioma.4.Before and after LFU sonication and BK infusion individually or in combination,RT-PCR were used to detect the protein expression levels of TJ associated proteins,elaudin-5,occludin,and ZO-1,in BTB model in vivo in rats with C6 brain glioma.5.Before and after LFU sonication and BK infusion individually or in combination,immunohistochemistry was performed to detect the distribution and expression of TJ associated proteins,claudin-5,occludin,and ZO-1,in BTB model in vivo in rats with C6 brain glioma. 6.Before and after LFU sonication and BK infusion individually or in combination,immunofluorescence was used to detect the distribution and expression of TJ associated proteins,claudin-5,occludin,and ZO-1,in BTB model in vivo in rats with C6 brain glioma.7.Before and after LFU sonication and BK infusion individually or in combination,Western blot and RT-PCR were performed to detect the protein expression levels of TJ associated proteins,ZO-1,occludin,and claudin-5,in BTB model in vivo in rats with C6 brain glioma.Results1.In C6 glioma rats,the permeability of BTB increased after LFU sonication, reached the peak at 1.5 h,and recovered 12 h later.2.In C6 glioma rats,TJ was opened by LFU,and the apoptosis of C6 glioma cells was induced,without neuronal injury.3.In C6 glioma rats,mRNA expression of TJ related proteins,claudin-5, occludin,and ZO-1,significantly decreased,reached the minimum at 1.5 h,and recovered 12 h later.4.In C6 glioma rats,protein expression of TJ related proteins,claudin-5, occludin,and ZO-1,significantly decreased,reached the minimum at 1.5 h,and recovered 12 h later.5.The permeability of BTB significantly increased after the individual application of LFU and BK,and further increased after LFU applied in combination with BK.6.TJ opening was observed under transmission electron microscope after LFU and BK applied individually and in combination.7.The mRNA expression of TJ related proteins,claudin-5,occludin,and ZO-1, significantly decreased after the application of LFU and BK individually,and further decreased after LFU applied in combination with BK.8.The protein expression of TJ related proteins,claudin-5,occludin,and ZO-1, significantly decreased after the application of LFU and BK individually,and further decreased after LFU applied in combination with BK. DiscussionIn the study,we proved that LFU can increase the permeability of BTB and open the BTB reversibly.No neuronal injury was observed,but the apoptosis of C6 glioma cells was induced during the sonication.The permeability of BTB significantly increased and TJ opening of BTB was observed by the combination of LFU and BK. Additionally,the mRNA and protein levels of TJ-related proteins,claudin-5,occludin, and ZO-1,reduced significantly.Furthermore,the permeability of BTB significantly increased and the expressions of TJ-related proteins,claudin-5,occludin,and ZO-1, significantly decreased in 5/6LFU+2/3BK and LFU+BK groups compared with LFU and BK groups.The results in this study indicate that the combination of LFU and low-dose BK increase the permeability of BTB in a synergistic manner in the rat model of C6 glioma,and TJ opening may contribute to the LFU/BK-induced increase in BTB permeability.The low frequency（1 MHz） and low pressure（0.48 MPa） ultrasound we used here is the same as that used in our previous study.The increased EB content after LFU sonication,indicating an increase in BTB permeability,confirmed our previous results. Different powers and pressures of LFU were also reported to open BBB locally and increase the permeability of agents and antibodies through BBB.Targeted delivery of doxorubicin and gadolinium into the brain was successfully enhanced by LFU with a frequency of 1.5-1.7 MHz and pressure amplitudes of 0.67-0.8 MPa,without a significant increase in brain temperature.However,brain tissue necrosis,neuron apoptosis,and red blood cell extravasation in the sonicated area have been reported after LFU sonication at relatively high pressures and frequencies（frequency of 690 kHz and pressure amplitude of 2.3 to 3.1 MPa or a frequency of 2.04 MHz and pressure amplitude of 0.3 to 2.3 MPa） by Hynynen et al.and McDannold et al.The low frequency and low pressure used in this study,which achieved a similar effect on BTB to other studies without detectable neuron damage,might reduce the adverse effect of LFU sonication and be applied clinically as a feasible approach in tumor treatment.The exact mechanism for LFU-induced increase of BTB permeability is unclear. LFU may alter BBB permeability by modifying cerebral blood flow through vasoconstriction,or by inertial cavitation.Besides,TJ is a stable structure to keep dynamic equilibrium of paracellular permeability and may be disorganized and reorganized quickly by extracellular stimulations.In this study,openings of TJ were found between brain microvessel endothelial cells and tumor tissues in all experimental groups,indicating that TJ opening is indeed involved in the increase of BTB permeability and the paraendothelial route may be one of the mechanisms for the increase of BTB permeability by LFU,as suggested in Hynynen et al.The reduction in TJ-related proteins,claudin-5,occludin,and ZO-1,in brain microvessels after LFU sonication confirmed the important role of TJ opening in BTB permeability.The increase in BTB permeability and down-regulation of TJ-related proteins after low-dose BK infusion are consistent with our previous study,indicating a paraendothelial route by BK.Other mechanisms may also be involved,such as the opening of ATP-activated(K_ATP) or Ca²⁺-activated(K_Ca) potassium channels.Despite the obvious effect of low-dose BK on BTB permeability,the duration of BK-induced increase in BTB permeability is usually transient,with the peak at 15 to 30 min and full recovery within 60 min.The clinical application of BK as a BTB opening substance is limited partly because of its transient effect.Since individual usage of either LFU or BK has noticeable limitations,we investigated the combinatory effect of LFU and low-dose BK in this study.To our knowledge,the combination of LFU and low-dose BK has not been reported.Both 5/6LFU+2/3BK and LFU+BK treatment induced a more potent increase in BTB permeability and reduction in ZO-1,occludin,and claudin-5 expressions than LFU and BK individually.Therefore,we conclude that LFU and BK act in a synergistic manner to increase the BTB permeability when used in combination.Furthermore,as there was no significant difference between 5/6LFU+2/3BK and LFU+BK groups,the dose of 5/6LFU+2/3BK may be used as an optimal dose for this combined treatment.It is likely that the low-dose combination may lower the currently reported side effects of LFU and BK individually.In addition,the considerably prolonged duration on the increase of BTB permeability by 5/6LFU+2/3BK（see results section） also favours a wider use of the combined application of LFU and BK.In summary,this study demonstrates that the combination of LFU and BK at low dose exerts a synergistic effect on increasing BTB permeability,possibly through a paraendothelial route.This finding may provide some new guidance to find a clinically applicable method to locally open BTB and deliver anti-tumor agents to tumors.Conclusions1.The optimum parameters for LFU to open BTB are frequency of 1 MHz, power of 12 mW,pressure of 0.48 MPa and exposure time of 20 s.2.LFU can increase the permeability of BTB and open BTB reversibly,inducing the apoptosis of C6 glioma cells and doing no harm to neurons.3.LFU can reduce the expression of TJ related proteins of claudin-5,occludin and ZO-1,at mRNA and protein levels,and TJ opening may be related to the effect induced by LFU.4.LFU and BK increase the permeability of BTB in a synergistic manner in the rat model of C6 glioma,and TJ opening may contribute to the LFU/BK-induced increase in BTB permeability.5.There was a further decrease in TJ related proteins of claudin-5,occludin,and ZO-1 reduced by LFU and BK in a synergistic manner.更多还原