Transcytosis of protein through the mammalian cerebral epithelium and endothelium .3. Receptor-mediated transcytosis through the blood-brain barrier of blood-borne transferrin and antibody against the transferrin receptor
Transcytosis of protein through the mammalian cerebral epithelium and endothelium .3. Receptor-mediated transcytosis through the blood-brain barrier of blood-borne transferrin and antibody against the transferrin receptor
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DOI：
10.1006/exnr.1996.0178
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发表时间：
1996-11-01
影响因子：
5.3
通讯作者：
Villegas, JC
中科院分区：
文献类型：
--
作者：
Broadwell, RD;BakerCairns, BJ;Villegas, JC
二铁-转铁蛋白(Tf；80K-WT.)和OX26抗体(150K-WT.)在光和超微结构水平上评价了天然辣根过氧化物酶(40K-Mel)作为血液到脑的跨细胞转移(跨细胞)的潜在载体在大鼠中的作用。WT)，它本身不会越过血脑屏障(BBB)。OX26，OX26的Fab片段(50K-Wt.)，Tf与两个铁离子以1：1的摩尔比不可逆地偶联到HRP上。以OX26为单抗的间接免疫过氧化物酶技术应用于冰冻切片表面或静脉注射给活体大鼠，在血脑屏障的毛细血管、小动脉和小静脉上发现TFR，而在供应室周器官的非血脑屏障血管(正中隆起、脉络丛)上则没有TFR。OX26-HRP和OX26(FAB)-HRP静脉注射和Diferric-Tf-HRP注射到颈动脉标记的中枢神经系统的血脑屏障血管，没有明显的血脑屏障破坏或血液探针渗入脑实质，在血脑屏障缺乏的部位没有观察到探针的反应产物。每个大分子偶联物都被血脑屏障内皮细胞吞噬，并被标记为假定的内吞泡泡、内小体和致密小体。OX26-HRP和Tf-HRP，而不是OX26(Fab)-HRP，似乎通过血脑屏障内皮细胞进行跨细胞反应，以随后标记血管周围细胞。OX26-HRP和Tf-HRP在细胞内和细胞外的分布明显不同：(1)血源性OX26-HRP在血脑屏障内皮细胞内的吞噬和隔离作用比Diferric-Tf-HRP更显著，(2)只有OX26-HRP标记了BBB内皮细胞的凝胶复合体，(3)注射Diferic-Tf-HRP的大鼠中枢神经系统血管周裂隙和血管周围细胞的过氧化物酶标记在注射后不到1h就明显，而血源性OX26-HRP的大鼠在注射后5min到6h内没有明显的过氧化物酶标记；(4)注射Diferic-Tf-HRP的大鼠中枢神经系统神经元和神经胶质细胞易于观察到过氧化物酶标记。结果表明，受体介导的Tf-HRP从血液到脑内皮细胞的转移比OX26-HRP更有效和直接。用血源性OX26-HRP标记血脑屏障内皮细胞的凝胶复合体表明，OX26-HRP的内皮转移遵循与吸附跨细胞过程相关的内皮内途径。提供了一张示意图，描绘了血脑屏障内皮细胞内的可能的细胞内和跨细胞路径，作为载体，血液传播的Diferric-Tf和OX26可将非脂溶的大分子运送到中枢神经系统，否则这些大分子将被血脑液屏障拒绝进入。(C)1996年学术出版社。
Diferric-transferrin (Tf; 80K mel. wt.) and the OX26 antibody (150K mel. 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 mel. wt.), which by itself does not cross the blood-brain barrier (BBB). OX26, the Fab fragment of OX26 (50K mel. 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 Gels 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 Gels 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. (C) 1996 Academic Press, Inc.