脑胶质瘤动物模型中血脑屏障紧密连接的变化

Changes of tight junctions between the brain microvascular endothelial cells in rats bearing C6 glioma

目的探讨脑胶质瘤对血脑屏障内皮细胞间紧密连接的影响及其分子机制。方法60只Wistar大鼠采用随机数字表法分为正常组（n=30）和脑肿瘤组（n＝30），脑肿瘤组按常规方法制作C6脑胶质瘤模型，造模后第21天行MRI检查后，取正常脑组织、肿瘤中心、肿瘤边缘及肿瘤远隔部位（边缘外2mm以上1组织，采用透射电镜观察两组大鼠血脑屏障紧密连接的超微结构特征，应用免疫组织化学染色和RT．PCR分别检测脑组织中紧密连接蛋白Claudin-5和Claudin-5 mRNA表达的变化。结果C6肿瘤细胞接种21d后，MRI可见大鼠右脑形成肿瘤。在透射电镜下，正常脑组织微血管相邻内皮细胞间可见连续条带状的紧密连接，细胞间未见裂隙；在肿瘤中心组织，仅有22．23％微血管相邻内皮细胞间可见条带状紧密连接，其余内皮细胞间可见明显裂隙，部分内皮细胞间连接为缝隙连接；在肿瘤边缘组织中，有57．15％微血管内皮细胞间存在紧密连接，其余内皮细胞间局部可见裂隙，部分内皮细胞问可见缝隙连接。免疫组化染色显示正常脑组织微血管内皮细胞Claudin－5呈强阳性表达：肿瘤中心组织微血管内皮细胞Claudin－5表达呈阴性；肿瘤边缘微血管内皮细胞Claudin－5呈弱阳性表达。RT—PCR结果示肿瘤中心Claudin－5 mRNA表达较肿瘤边缘、肿瘤远隔部位、正常组脑组织降低，差异均有统计学意义俨均〈0．05）。结论在胶质瘤的发生、发展过程中．胶质瘤细胞可以导致血脑屏障内皮细胞紧密连接蛋白Claudin－5的表达下降及血脑屏障紧密连接结构的破坏。

Objective To investigate the changes in the tight junctions of the blood-brain barrier （BBB） in a rat model bearing C6 glioma. Methods Rat models bearing C6 glioma were established in 30 male Wistar rats by stereotactic injection of C6 glioma cells into the caudate nuclei, with another 30 rats as the normal control group. Twenty-one days after the injection, magnetic resonance imaging （MRI） was performed, and the normal brain tissue, tumor core, tumor margin and the ipsilateral hemisphere tissues 2 rnm from the tumor margin were sampled for ultrastructural observation of the BBB using electron microscopy. Immunohistochemistry and RT-PCR were used to analyze the expression of claudin-5 protein and mRNA in these tissues, respectively. Results MRI revealed tumor formation in the brain 21 days after C6 cell injection. In normal control brain tissues, the paracellular cleft between the adjacent endothelial cells was sealed by continuous tight junction strands, which were found in only 22.23% of the microvesscls in the core of the brain gliomas, and obvious paracellular clefts were found between the adjacent endothelial cells in other microvessels. In the tissues on the tumor margin, intercellular tight junctions were found in 57.15% of the microvessels with the rest microvessels having obvious paracellular clefts. Immunohistochemistry showed strong claudin-5 positivity in the control brain tissue but yielded negative results in the core of the gliomas. Compared with the core of the gliomas, the tissues on the tumor margin, those 2 mm from the tumor margin and the control brain tissues showed significantly increased claudin-5 expression （P〈0.05,respectively）. Conclusion In C6 glioma-bearing rats, the continuity of the tight junctions in the BBB is interrupted due to decreased expression of elaudin-5 in the brain gliomas.