超声微泡促进骨髓间质细胞跨血脑屏障迁移的实验研究
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摘要
缺血性中风是致死、致残的主要病因之一。脑损伤很容易遗留永久性神经功能缺失，其原因体现在脑组织对自身的损伤修复能力不强，同时采用药物治疗效果不佳。研究学者通过动物实验以及临床实验证明：移植胚胎神经细胞和多能干细胞可促进损伤脑组织的修复，改善神经功能缺失症状，提示细胞移植方法是治疗缺血脑损伤的很有希望的途径。大量实验研究表明,骨髓间质细胞(bone marrow stromal cells, BMSC)是最有潜力的一种修复细胞，其在脑内移植后，能够存活下来，并不断迁移和分化，而后在一定程度上修复脑梗死后神经功能的障碍。移植细胞促进脑损伤修复的机制包括替代坏死细胞功能、抑制局部瘢痕形成和促进损伤局部神经再生等，而移植细胞向脑损伤局部的迁移是促进脑损伤修复的前提。因此，探讨促进移植细胞向脑损伤局部迁移的措施有非常重要的实际意义。
     血脑屏障影响经静脉移植细胞向脑损伤局部的迁移，降低移植治疗效果。近年研究发现，超声联合微泡技术可安全、有效地暂时性开放血脑屏障，促进治疗药物通过血脑屏障，提高脑实质内药物浓度，达到提高治疗效果的目的。但目前尚未见到超声联合微泡技术对移植细胞向脑内迁移影响的研究报道。因此，深入探讨超声联合微泡技术促进经静脉移植骨髓间质细胞向脑内迁移的可能性及其机制，有望为缺血性中风带来新的治疗途径。
     研究内容：(1)评价大鼠前脑缺血/再灌注模型血脑屏障完整性：光镜和电镜下观察模型动物血脑屏障完整性：包括脑血管内皮细胞紧密连接、基底膜和星形胶质细胞伪足完整性；观察血脑屏障结构蛋白表达：包括脑血管内皮细胞；闭合蛋白claudin-5基底膜；IV胶原、层连蛋白星型胶质细胞突起；S100B蛋白等；评价血脑屏障功能：光镜观察伊文思蓝渗出量，电镜下观察硝酸镧染色。(2)观察超声联合微泡技术对前脑缺血/再灌注大鼠血脑屏障通透性的影响：(3)观察超声联合微泡技术能否影响经静脉移植的骨髓间质细胞向缺血性脑损伤区迁移的活动：观察体外扩增和鉴定骨髓间质细胞；观察经静脉注射移植骨髓间质细胞；不比较骨髓间质细胞向脑缺血损伤区迁移的数量和距离；观察AchE阳性纤维密度。(4)观察超声联合微泡技术对经静脉移植的骨髓间质细胞对大鼠神经功能的影响:水迷宫试验观察空间学习、记忆能力。
     研究结果：(1)缺血再灌注模型可以在一定程度上增加血脑屏障通透性。(2)超声微泡可以开放脑缺血/再灌注血脑屏障。(3)超声微泡可以有效开放BBB，并促进MSCS向脑内迁移和AchE阳性纤维增多。(4)超声微泡联合MSCs移植可以促进前脑缺血大鼠空间学习和记忆能力。
     研究结论：超声微泡可以显著促进骨髓间充质细胞向脑内的迁移，进而促进前脑缺血大鼠行为能力的显著提高，MSCs颅内移植可以改善前脑缺血大鼠的空间学习能力，对记忆能力影响小。MSCs取材简单，扩增迅速，来源丰富，MSCs移植治疗脑缺血具有广阔的临床应用前景。但超声微泡开放血脑屏障的机制和MSCs颅内移植移植促进神经功能改善的机制尚需要进一步的探索。
Ischemic stroke is one of the main causes of death and disability. Because the braintissue has the limited self-repair ability, drug treatment can not get ideal effect. Therefore,the people often left permanent neurological function deficiency.
     Animal experiment and clinical study found that transplantation of embryonic neuralcells and pluripotent stem cells could promote the repair of damaged brain tissue, andimprove neurological deficit symptoms, which suggested that cell transplantation for thetreatment of ischemic cerebral injury is a very promising way. Lots of experiments showthat the bone marrow stromal cells (bone marrow stromal cells, BMSC) transplantationcould migrate, differentiate, and survive in the host brain, which could promote therecovery of neural function after cerebral infarction. The mechanism of transplanted cellspromoted brain damage repair including alternative function of necrotic cells, inhibitingscar and promote local damage nerve regeneration. The local migration of transplanted cellsto the brain damage is a prerequisite for promoting the repair of brain injury. Therefore, themethod to promote local migration of transplanted cells into the brain injury is veryimportant.
     The blood brain barrier after vein transplantation to brain injury local migration reducethe treatment effect of transplantation. In recent years, the study found that ultrasoundcombined with microbubble technology could temporary open the blood brain barrier safelyand effectively. The technology could promote the drug through the blood brain barrier andimprove brain drug concentration to improve the treatment effect. But ultrasound combinedwith microbubble on the migration of transplanted cells in the brain has not been studied.Therefore, the study of ultrasound combined with microbubble technology transplantationof intravenous bone marrow stromal cells into brain migration is expected to bring a newway for treatment of ischemic stroke.
     Methods:(1) The evaluation of forebrain ischemia/reperfusion rat model BBBintegrity. Observation of animal model of blood brain barrier integrity in the lightmicroscope and electron microscope, including brain vascular endothelial cell tight junction,basement membrane and glial cell pseudopodia integrity. Observation of the blood brainbarrier structure protein expression, including brain vascular endothelial cells, closedprotein claudin-5basement membrane, collagen IV, laminin astrocyte processes, S100Bprotein. Evaluating blood-brain barrier function of Evans blue extravasation. Observationof light microscope, electron microscope observation of lanthanum nitrate staining.(2)Observing the effects of ultrasound combined with microbubble technology on forebrainischemia/reperfusion blood-brain barrier permeability in rats.(3) Observing the effect ofultrasound combined with microbubble technology on intravenous transplantation of bonemarrow stromal cells into ischemic brain damage zone migration. in vitro amplification andidentification of bone marrow stromal cells; intravenous transplantation of bone marrowstromal cells; bone marrow stromal cells do not compare to cerebral ischemia area to movethe number and distance shift; observation the density of AchE positive fibers.(4)Observing the ultrasound combined with microbubble technology on intravenoustransplantation of bone marrow stromal cells on neural function in rats with water maze testof spatial learning and memory ability.
     Results:(1) Ischemia reperfusion model can increase the permeability of blood brainbarrier in some degree.(2)The ultrasound microbubble can open cerebral ischemia/reperfusion blood-brain barrier.(3)The ultrasound microbubble can open BBB, andpromote MSCS migration to the brain and increase AchE positive fibers.(4)The ultrasonicmicrobubbles combined with MSCs transplantation can promote the learning and memoryability of forebrain ischemia rats.
     Conclusion: The blood of ultrasound microbubbles could promote bone marrowmesenchymal cells to intracranial migration, and improve the behavior of forebrainischemia rat ability, MSCs intracranial transplantation could improve cerebral ischemia ratspatial learning ability, but could not improve memory ability. MSCs could be easy get andrapid amplificated, MSCs transplantation for treatment of cerebral ischemia have broadprospects in clinical application. The mechanism of MSCs intracranial transplantation ofultrasound microbubble opening blood brain barrier to promote nerve function improvement still needs be further studied.
引文
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