CAJ | 학술논문

目的：建立一种纯度和活力较高、简单实用的大鼠脑微血管内皮细胞分离及原代培养方法,为建立体外血脑屏障提供材料。方法采集1-2周SD大鼠大脑皮质,应用Ⅱ型胶原酶和分散酶/胶原酶连续消化法,筛网过滤法及20%BSA和44%Percoll两次梯度离心法获得脑微血管段后,接种于培养瓶中进行原代培养,采用倒置显微镜对所培养的细胞进行形态学观察,以Ⅷ因子相关抗原免疫染色法对其进行鉴定。结果体外培养2h后,微血管内皮细胞爬出血管段进行贴壁生长,3~4 d呈典型的铺路卵石样结构,Ⅷ因子相关抗原免疫组化检测内皮细胞表达呈阳性,可见细胞胞质呈棕色,阳性细胞占99%以上。结论该方法能成功地分离并培养出高纯度的大鼠脑微血管内皮细胞,对体外血脑屏障的建立以及脑微血管内皮细胞的生物学特性和功能的深入研究具有重要意义。
목적：건립일충순도화활력교고、간단실용적대서뇌미혈관내피세포분리급원대배양방법,위건입체외혈뇌병장제공재료。방법채집1-2주SD대서대뇌피질,응용Ⅱ형효원매화분산매/효원매련속소화법,사망과려법급20%BSA화44%Percoll량차제도리심법획득뇌미혈관단후,접충우배양병중진행원대배양,채용도치현미경대소배양적세포진행형태학관찰,이Ⅷ인자상관항원면역염색법대기진행감정。결과체외배양2h후,미혈관내피세포파출혈관단진행첩벽생장,3~4 d정전형적포로란석양결구,Ⅷ인자상관항원면역조화검측내피세포표체정양성,가견세포포질정종색,양성세포점99%이상。결론해방법능성공지분리병배양출고순도적대서뇌미혈관내피세포,대체외혈뇌병장적건립이급뇌미혈관내피세포적생물학특성화공능적심입연구구유중요의의。
Aim To establish a highly purified,active and prac-tical extract and primitive culture method for rat brain microvas-cular endothelial cells ( BMECs) for providing materials for con-struction in vitro blood-brain barrier ( BBB) model. Methods Cerebral cortex of 1-2 week SD rats was collected,and successive digestion with typeⅡcollagenase and collagenase/dispase, sieve filtration and then twice gradient centrifugation in 20% BSA and 44% Percoll condition were used to obtain brain microvascular section. After that brain microvascular section was seeded in cul-ture bottle and then primarily cultured. Inverted microscope and factor-VIII relative antigen immunostaining methods were used for cellular morphological observation and identification. Results BMECs climbed out the vessel segment and proliferated with ad-herence after 2 h in vitro culture,and they became typical peb-bles structure after further 3-4 d culture. Morever, factor-VIII relative antigen immunostaining identified that expression for the endothelial cells was positive,cytoplasm was brown and positive cells account for more than 99%. Conclusion Rat BMECs with high purity could be extracted and cultured by using the above methods,and it has great potential for construction in vitro BBB model and in depth studies on biological characteristics and func-tions of BMECs.