Broadwell R D, Baker-Cairns B J, Friden P M, Oliver C, Villegas J C
Office of Research Integrity, U.S. Public Health Service, Rockville, Maryland 20852, USA.
Exp Neurol. 1996 Nov;142(1):47-65. doi: 10.1006/exnr.1996.0178.
Diferric-transferrin (Tf; 80K mol. wt.) and the OX26 antibody (150K mol. wt.) against the transferrin receptor (TfR) were evaluated in the rat at light and ultrastructural levels as potential vehicles for the blood to brain transcellular transfer (transcytosis) of native horseradish peroxidase (40K mol. wt.), which by itself does not cross the blood-brain barrier (BBB). OX26, the Fab fragment of OX26 (50K mol. wt.), and Tf complexed to two ferric ions were conjugated to HRP irreversibly in a 1:1 molar ratio. The indirect immunoperoxidase technique with OX26 as the monoclonal primary antibody applied to the surface of cryostat sections or delivered intravenously to the live rat revealed TfRs on BBB capillaries, arterioles, and venules; TfRs were absent on non-BBB vessels supplying the circumventricular organs (i.e., median eminence, choroid plexus). OX26-HRP and OX26(Fab)-HRP delivered intravenously and diferric-Tf-HRP administered into the carotid artery labeled BBB vessels throughout the CNS without discernible disruption of the BBB or extravasation of the blood-borne probes into the brain parenchyma. No reaction product for the probes was observed in sites deficient in a BBB. Each of the macromolecular conjugates was endocytosed by BBB endothelia and labeled presumptive endocytic vesicles, endosomes, and dense bodies. OX26-HRP and Tf-HRP, but not OX26(Fab)-HRP, appeared to undergo transcytosis through BBB endothelia for subsequent labeling of perivascular cells. Distinct differences in the intracellular and extracellular distributions between OX26-HRP and Tf-HRP were identified: (1) endocytosis and sequestration of blood-borne OX26-HRP within BBB endothelia were more prominent than those for diferric-Tf-HRP; (2) only OX26-HRP labeled the Golgi complex in BBB endothelia; (3) peroxidase labeling of CNS perivascular clefts and perivascular cells in rats receiving diferric-Tf-HRP was conspicuous at less than 1 h postinjection but not so in rats with blood-borne OX26-HRP at 5 min through 6 h postinjection; and (4) peroxidase-labeled CNS neurons and glial cells were identified readily in rats receiving diferric-Tf-HRP. The results suggest that the receptor-mediated, transendothelial transfer of Tf-HRP from blood to brain is more efficient and direct than that of OX26-HRP. Labeling of the Golgi complex in BBB endothelia with blood-borne OX26-HRP implies that the transendothelial transfer of OX26-HRP follows intraendothelial pathways associated with the process of adsorptive transcytosis. A diagram is provided depicting the possible intracellular and transcellular pathways within BBB endothelia available to blood-borne diferric-Tf and OX26 as vectors for delivery into the CNS of non-lipid-soluble macromolecules that otherwise are denied entry by the blood-brain fluid barriers.
在大鼠体内，对双铁转铁蛋白（Tf；分子量80K）和抗转铁蛋白受体（TfR）的OX26抗体（分子量150K）进行了光镜和超微结构水平的评估，以确定它们作为天然辣根过氧化物酶（分子量40K）从血液到脑跨细胞转运（转胞吞作用）潜在载体的可能性，辣根过氧化物酶本身无法穿过血脑屏障（BBB）。OX26、OX26的Fab片段（分子量50K）以及与两个铁离子络合的Tf以1:1的摩尔比不可逆地与HRP偶联。以OX26作为单克隆一抗，采用间接免疫过氧化物酶技术应用于冷冻切片表面或静脉注射给活体大鼠，结果显示BBB毛细血管、小动脉和小静脉上存在TfR；供应室周器官（即正中隆起、脉络丛）的非BBB血管上不存在TfR。静脉注射的OX26-HRP和OX26(Fab)-HRP以及颈动脉注射的双铁-Tf-HRP标记了整个中枢神经系统的BBB血管，且未观察到BBB有明显破坏或血源探针渗入脑实质。在缺乏BBB的部位未观察到探针的反应产物。每种大分子偶联物均被BBB内皮细胞内吞，并标记了假定的内吞小泡、内体和致密小体。OX26-HRP和Tf-HRP似乎通过BBB内皮细胞进行转胞吞作用，随后标记血管周围细胞，但OX26(Fab)-HRP未出现这种情况。确定了OX26-HRP和Tf-HRP在细胞内和细胞外分布上的明显差异：（1）血源OX26-HRP在BBB内皮细胞内的内吞和隔离比双铁-Tf-HRP更显著；（2）只有OX26-HRP标记了BBB内皮细胞中的高尔基体；（3）注射双铁-Tf-HRP的大鼠在注射后不到1小时，中枢神经系统血管周围间隙和血管周围细胞的过氧化物酶标记明显，但注射血源OX26-HRP的大鼠在注射后5分钟至6小时未出现这种情况；（4）在接受双铁-Tf-HRP的大鼠中很容易识别出过氧化物酶标记的中枢神经系统神经元和神经胶质细胞。结果表明，Tf-HRP从血液到脑的受体介导的跨内皮转运比OX26-HRP更有效、更直接。血源OX26-HRP对BBB内皮细胞中高尔基体的标记表明，OX26-HRP的跨内皮转运遵循与吸附性转胞吞作用相关的内皮内途径。提供了一张示意图，描绘了血源双铁-Tf和OX26作为载体，在BBB内皮细胞内可能的细胞内和跨细胞途径，用于将非脂溶性大分子输送到中枢神经系统，否则这些大分子会被血脑液屏障阻止进入。
