犬脐静脉血管内皮细胞的分离培养与鉴定_《中国修复重建外科杂志》

作者：

胡泉 , 柴家科 , 刘玲英 , 侯玉森 , 王一贺 , 李百玲 , 杨红明

解放军总医院第一附属医院烧伤研究所（北京，100048）;

关键词：

血管内皮细胞脐静脉细胞培养细胞鉴定犬

DOI：

10.7507/1002-1892.20130105

视频：

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目的建立一种简便、高效的犬脐静脉血管内皮细胞分离方法，并进行培养和鉴定。方法无菌条件下取12只新生比格犬脐带共12根，脐带长（13.0 ± 1.5）cm。PBS液冲洗后，各取4根脐带注入1%Ⅰ型胶原酶分别消化脐静脉5、7、10 min后，收集细胞并行锥虫蓝计数；倒置相差显微镜下观察细胞形态，待细胞爬满培养瓶后以1∶2比例传代。取Ⅰ型胶原酶消化7 min获得的第3代细胞，通过免疫荧光染色和流式细胞仪检测细胞中血管假性血友病因子（von Willebrand factor，vWF）和血小板-内皮细胞黏附分子CD31表达，进行细胞鉴定；MTT检测细胞增殖。结果消化5、10 min后未收集到或偶尔收集到血管内皮细胞；消化7 min可见大量血管内皮细胞，培养24 h后细胞呈多角形，随培养时间延长细胞逐渐呈“铺路石”样生长，细胞核较大，多为单核，少数细胞为双核，传代后细胞形态无明显变化。荧光倒置显微镜下观察，培养细胞vWF和CD31表达阳性；流式细胞仪检测示培养细胞 vWF阳性表达率为99.0% ± 0.7%，CD31阳性表达率为98.0% ± 1.2%，提示培养细胞为血管内皮细胞。MTT检测示，随培养时间延长，血管内皮细胞增殖速度明显提高。结论采用酶消化法从犬脐静脉中分离培养血管内皮细胞操作简便，且细胞数量多、纯度高。

引用本文： 胡泉,柴家科,刘玲英,侯玉森,王一贺,李百玲,杨红明. 犬脐静脉血管内皮细胞的分离培养与鉴定. 中国修复重建外科杂志, 2013, 27(4): 460-463. doi: 10.7507/1002-1892.20130105 复制

1.	Shupp JW, Nasabzadeh TJ, Rosenthal DS, et al. A review of the local pathophysiologic bases of burn wound progression. J Burn Care Res, 2010, 31(6): 849-873.
2.	Pries AR, Kuebler WM. Normal endothelium. Handb Exp Pharmacol, 2006, (176 Pt 1): 1-40.
3.	胡静, 仲照东, 邹萍. 血管内皮细胞的生物学特性. 国际输血及血液学杂志, 2010, 33(1): 76-79.
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6.	Siow RC. Culture of human endothelial cells from umbilical veins. Methods Mol Biol, 2012, 806: 265-274.
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8.	Baudin B, Bruneel A, Bosselut N, et al. A protocol for isolation and culture of human umbilical vein endothelial cells. Nat Protoc, 2007, 2(3): 481-485.
9.	Jaffe EA, Nachman RL, Becker CG, et al. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest, 1973, 52(11): 2745-2756.
10.	Geerts WJ, Vocking K, Schoonen N, et al. Cobblestone HUVECs: a human model system for studying primary ciliogenesis. J Struct Biol, 2011, 176(3): 350-359.
11.	黄起壬, 戴育成, 李剑, 等. 三种人脐静脉内皮细胞分离方法的比较. 江西医学院学报, 2005, 45(5): 8-14.
12.	Fehrenbach ML, Cao G, Williams JT, et al. Isolation of murine lung endothelial cells. Am J Physiol Lung Cell Mol Physiol, 2009, 296(6): L1096-1103.
13.	Jin Y, Liu Y, Antonyak M, et al. Isolation and characterization of vascular endothelial cells from murine heart and lung. Methods Mol Biol, 2012, 843: 147-154.
14.	张臣, 李彤, 侯跃龙, 等. 人脐静脉血管内皮细胞的高效分离与培养. 天津医药, 2010, 38(7): 605-607.
15.	Winiarski BK, Acheson N, Gutowski NJ. An improved and reliable method for isolation of microvascular endothelial cells from human omentum. Microcirculation, 2011, 18(8): 635-645.
16.	刘长乐, 郑心田, 李广平, 等. 体外人脐静脉内皮细胞的培养鉴定及抗原表达分析. 山东医药, 2011, 51(36): 25-26.
17.	Lip GY, Blann A. von Willebrand factor: a marker of endothelial dysfunction in vascular disorders? Cardiovasc Res, 1997, 34(2): 255-265.
18.	Horvath B, Hegedus D, Szapary L, et al. Measurement of von Willebrand factor as the marker of endothelial dysfunction in vascular diseases. Exp Clin Cardiol, 2004, 9(1): 31-34.
19.	Janeczek Portalska K, Leferink A, Groen N, et al. Endothelial differentiation of mesenchymal stromal cells. PLoS One, 2012, 7(10): e46842.
20.	Sobczak M, Dargatz J, Chrzanowska-Wodnicka M. Isolation and culture of pulmonary endothelial cells from neonatal mice. J Vis Exp, 2010, (46): doi: pii: 2316.

1. Shupp JW, Nasabzadeh TJ, Rosenthal DS, et al. A review of the local pathophysiologic bases of burn wound progression. J Burn Care Res, 2010, 31(6): 849-873.
2. Pries AR, Kuebler WM. Normal endothelium. Handb Exp Pharmacol, 2006, (176 Pt 1): 1-40.
3. 胡静, 仲照东, 邹萍. 血管内皮细胞的生物学特性. 国际输血及血液学杂志, 2010, 33(1): 76-79.
4. Aird WC. Endothelium in health and disease. Pharmacol Rep, 2008, 60(1): 139-143.
5. Marin V, Kaplanski G, Grès S, et al. Endothelial cell culture: protocol to obtain and cultivate human umbilical endothelial cells. J Immunol Methods, 2001, 254(1-2): 183-190.
6. Siow RC. Culture of human endothelial cells from umbilical veins. Methods Mol Biol, 2012, 806: 265-274.
7. Nachman RL, Jaffe EA. Endothelial cell culture: beginnings of modern vascular biology. J Clin Invest, 2004, 114(8): 1037-1040.
8. Baudin B, Bruneel A, Bosselut N, et al. A protocol for isolation and culture of human umbilical vein endothelial cells. Nat Protoc, 2007, 2(3): 481-485.
9. Jaffe EA, Nachman RL, Becker CG, et al. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest, 1973, 52(11): 2745-2756.
10. Geerts WJ, Vocking K, Schoonen N, et al. Cobblestone HUVECs: a human model system for studying primary ciliogenesis. J Struct Biol, 2011, 176(3): 350-359.
11. 黄起壬, 戴育成, 李剑, 等. 三种人脐静脉内皮细胞分离方法的比较. 江西医学院学报, 2005, 45(5): 8-14.
12. Fehrenbach ML, Cao G, Williams JT, et al. Isolation of murine lung endothelial cells. Am J Physiol Lung Cell Mol Physiol, 2009, 296(6): L1096-1103.
13. Jin Y, Liu Y, Antonyak M, et al. Isolation and characterization of vascular endothelial cells from murine heart and lung. Methods Mol Biol, 2012, 843: 147-154.
14. 张臣, 李彤, 侯跃龙, 等. 人脐静脉血管内皮细胞的高效分离与培养. 天津医药, 2010, 38(7): 605-607.
15. Winiarski BK, Acheson N, Gutowski NJ. An improved and reliable method for isolation of microvascular endothelial cells from human omentum. Microcirculation, 2011, 18(8): 635-645.
16. 刘长乐, 郑心田, 李广平, 等. 体外人脐静脉内皮细胞的培养鉴定及抗原表达分析. 山东医药, 2011, 51(36): 25-26.
17. Lip GY, Blann A. von Willebrand factor: a marker of endothelial dysfunction in vascular disorders? Cardiovasc Res, 1997, 34(2): 255-265.
18. Horvath B, Hegedus D, Szapary L, et al. Measurement of von Willebrand factor as the marker of endothelial dysfunction in vascular diseases. Exp Clin Cardiol, 2004, 9(1): 31-34.
19. Janeczek Portalska K, Leferink A, Groen N, et al. Endothelial differentiation of mesenchymal stromal cells. PLoS One, 2012, 7(10): e46842.
20. Sobczak M, Dargatz J, Chrzanowska-Wodnicka M. Isolation and culture of pulmonary endothelial cells from neonatal mice. J Vis Exp, 2010, (46): doi: pii: 2316.

《中国修复重建外科杂志》

犬脐静脉血管内皮细胞的分离培养与鉴定

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《中国修复重建外科杂志》

犬脐静脉血管内皮细胞的分离培养与鉴定

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