大鼠大脑微血管片段的分离纯化及鉴定

ISOLATION,PURIFICATION AND IDENTIFICATION OF RAT BRAIN MICROVESSELS

目的 :分离大鼠大脑微血管并纯化去除完整的神经细胞 ,用于克隆血脑屏障上的特异表达基因。方法 :采用液相合成法制备粒径 2 0 0～ 5 0 0nm的铁氧体磁珠 ,经两侧颈内动脉插管注入大鼠大脑半球。采用机械分离和酶消化相结合的方法解离脑组织 ,用筛网滤去组织块和大血管 ,再在磁场下分选标记磁珠的微血管片段 ,并从形态学、分子生物学和生物活性角度鉴定获得的脑微血管片段。结果 :扫描电镜下没有发现微血管周围存在完整的神经细胞 ,但在部分区域有胶质的终足包裹。全脑组织微管相关蛋白 2a、谷氨酰胺合成酶和CD31的RT PCR产物均在相应位置出现阳性条带 ,分离纯化的脑微血管仅CD31阳性。微血管片段内皮细胞摄取的Rh12 3荧光强度显著低于传代培养的微血管内皮细胞荧光强度。结论 :采用本方法可以获得高纯度的、不附带完整神经细胞的脑微血管片段。

Aim: To isolate and purify the brain microvessels without intact neural cells used for cloning specific gene at the blood-brain-barrier. Methods: Magnetic beads ranging from 200～500 nm were synthesized and infused into cerebral spheres through carotid arteries.The brain tissues were dissected by machinetic and enzymatic methods,and sieved to discharge tissues and large blood vessels. The brain microvessels labelized by magnetic beads were sorted in magnetic fields,and identified by morphology,molecular biology and biologic activity. Results: Scanning electric micrograph of the obtained brain microvessels showed the vessels grossly free of adjoining neural cells except an occasional nerve ending. RT-PCR products of microtube-associated protein 2a,glutamine synthetase and CD31 from brain tissue had positive lanes,but only CD31 had positive lanes from isolated microvessels. The endothelial cells from isolated microvessels had more fluorescence than that from cultured endothelial cells. Conclusion: Highly purified microvessels without intact neural cells can be obtained by this method.