改良原代大鼠脑微血管内皮细胞的培养方法及鉴定

唐元瑜

doi:10.16098/j.issn.0529-1356.2020.01.024

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解剖学报 ›› 2020, Vol. 51 ›› Issue (1) : 135-138. DOI: 10.16098/j.issn.0529-1356.2020.01.024

技术方法

改良原代大鼠脑微血管内皮细胞的培养方法及鉴定

唐元瑜^1*马华根² 刘昭德³

作者信息 +

Improvement on the methods about the primary culture of rat brain microvascular endothelial cells and identification

TANG Yuan-yu^1* MA Hua-gen² LIU Zhao-de³

Author information +

文章历史 +

摘要

目的改良原代大鼠脑微血管内皮细胞分离培养方法。方法选取4～6周龄SD大鼠6只，经开颅取脑、漂洗剪碎、过筛、牛血清白蛋白密度梯度离心、Ⅱ型胶原酶及胶原酶-分散酶两次连续酶消化后进行原代培养。通过细胞形态学观察和第Ⅷ因子免疫细胞化学染色鉴定所培养的目的细胞。结果体外培养12～24 h后，细胞以贴壁的脑微血管段为中心，放射状向外周移行，并逐渐扩大成团簇状；细胞融合后则呈典型的单层、扁平、“铺路石样”镶嵌式排列。第Ⅷ因子免疫细胞化学染色检测，胞质呈棕红色，表达为阳性，阳性细胞率达99％以上。结论改良方法能够成功高效分离培养出原代大鼠脑微血管内皮细胞。

Abstract

Objective To improve the method about the primary culture of rat brain microvascular endothelial cells in vitro. Methods The SD rats aged from 4 to 6 weeks were chosen as research object. After craniotomy, washing and cutting, sieving, density gradient concentration of BSA, digestion of type Ⅱ collagenase and collagenase dispersive enzyme twice, the primary culture was carried out. The target cells were indentified by morphological abservation and immunocytochemical staining of facter Ⅷ. Results Cultured for 12 to 24 hours，the cells in vitro migrated outward from the microvascular section. The cells appeared polygonal-shaped，and proliferated in a clustered monolayer. the cell growth density reached 70％-80％ of the bottle bottom after 3 days，and arranged like cobbles. The correlation antigen of Ⅷ factor was positive，they reached confluence with over purity 99％. Conclusion The method is available that can successfully separate and cultivate microvascular endothelial cells of rat brains in vitro.

导出引用

唐元瑜马华根刘昭德. 改良原代大鼠脑微血管内皮细胞的培养方法及鉴定[J]. 解剖学报. 2020, 51(1): 135-138 https://doi.org/10.16098/j.issn.0529-1356.2020.01.024

TANG Yuan-yu MA Hua-gen LIU Zhao-de. Improvement on the methods about the primary culture of rat brain microvascular endothelial cells and identification[J]. Acta Anatomica Sinica. 2020, 51(1): 135-138 https://doi.org/10.16098/j.issn.0529-1356.2020.01.024

中图分类号： R322.81

参考文献

［1］ Panula P，Joo F，Rechardt L，et al. Evidence for the presence viable endothelial cells in cultures derived from dissociated rat brain［J］.Experientia，1978，34（1）：95-97.

［2］ Guo Y，Yi PF， LU ZhJ，et al.Culture and idendtification of rat brain microvascular endothelial cells in vitro［J］.Journal of Shenyang Agricultural University，2010，41（2）：170-174. (in Chinese)

郭洋，伊鹏霏，吕钊君，等.大鼠脑微血管内皮细胞的体外培养及鉴定［J］.沈阳农业大学学报，2010，41（2）：170-174.

［3］ Tian LY，Li ShF，Zhou D.Separation，culture and purification of rat brain microendothelial cell［J］.Si Chuan University Journal of Medical Science（Medical Science Edition），2010，41（5）：869-872. (in Chinese)

田林郁，李胜富，周东.大鼠脑微血管内皮细胞的分离、培养及不同纯化方法的比较［J］.四川大学学报(医学版)，2010，41（5）：869-872.

［4］ Ma HG，Tang YY，Ji LJ.Primary culture and identification of microvascular endothelial cells of rats［J］.Chinese Journal of Anatomy，2018，41（3）：276-279. (in Chinese)

马华根，唐元瑜，纪立金.原代大鼠脑微血管内皮细胞的培养及鉴定［J］.解剖学杂志，2018，41（3）：276-279.

［5］ Lu WB，Qin WW，Liu ShY，et al.Purified culture and identification of the rat brain microvascular endotheliar cells［J］. Chinese Journal of Cerebrovascular Diseases，2011，8（10）：535-538. (in Chinese)

鹿文葆，秦伟伟，刘淑英，等.大鼠脑微血管内皮细胞的纯化培养与鉴定［J］.中国脑血管病杂志，2011，8（10）：535-538.

［6］ Song XB，Fan ChE，Wang Y.Primary culture and identification of mouse brain microvascular endothelial cell［J］.Journal of Nanjing Normal University（Natural Science Edition），2017，40（3）：138-143. (in Chinese)

宋晓彬，范春娥，王烨.小鼠脑微血管内皮细胞的分离培养与鉴定［J］.南京师范大学报（自然科学版），2017，40（3）：138-143.

［7］ Zha YF，Zhang Sh， Su H，et al. A highly purified and active extract and primitive culture method for rat microvascular endothelial cells［J］.Chinese Pharmacological Bulletin，2014，30（11）：1616-1619. (in Chinese)

查雨锋，张顺，苏航，等.一种高纯度和高活力的大鼠脑微血管内皮细胞的提取及原代培养方法［J］.中国药理学通报，2014，30（11）：1616-1619.

［8］ Lou JN.Culture of microvascular endothelial cells and the application of medical research［J］. Chinese Journal of Microcirculation，2004，14（3）：5-8. (in Chinese)

娄晋宁.微血管内皮细胞的培养及其在医学研究中的应用［J］.微循环学杂志，2004，14（3）：5-8.

［9］ Zhan YL，Li WY，Myron Y.Separation，culture and appraisement of bovine choroidal microvascular endothelial cells［J］.Acta Anatomica Sinica，1997，28（3）：297-300. (in Chinese)

詹永乐，李维业，Myron Y.牛脉络膜微血管内皮细胞的分离培养和鉴定［J］.解剖学报，1997，28（3）：297-300.