犬臍靜脈血管內皮細胞的分離培養與鑒定_《中國修復重建外科雜志》

作者：

胡泉 , 柴家科 , 劉玲英 , 侯玉森 , 王一賀 , 李百玲 , 楊紅明

解放軍總醫院第一附屬醫院燒傷研究所（北京，100048）;

關鍵詞：

血管內皮細胞臍靜脈細胞培養細胞鑒定犬

DOI：

10.7507/1002-1892.20130105

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目的建立一種簡便、高效的犬臍靜脈血管內皮細胞分離方法，并進行培養和鑒定。方法無菌條件下取12只新生比格犬臍帶共12根，臍帶長（13.0 ± 1.5）cm。PBS液沖洗后，各取4根臍帶注入1%Ⅰ型膠原酶分別消化臍靜脈5、7、10 min后，收集細胞并行錐蟲藍計數；倒置相差顯微鏡下觀察細胞形態，待細胞爬滿培養瓶后以1∶2比例傳代。取Ⅰ型膠原酶消化7 min獲得的第3代細胞，通過免疫熒光染色和流式細胞儀檢測細胞中血管假性血友病因子（von Willebrand factor，vWF）和血小板-內皮細胞黏附分子CD31表達，進行細胞鑒定；MTT檢測細胞增殖。結果消化5、10 min后未收集到或偶爾收集到血管內皮細胞；消化7 min可見大量血管內皮細胞，培養24 h后細胞呈多角形，隨培養時間延長細胞逐漸呈“鋪路石”樣生長，細胞核較大，多為單核，少數細胞為雙核，傳代后細胞形態無明顯變化。熒光倒置顯微鏡下觀察，培養細胞vWF和CD31表達陽性；流式細胞儀檢測示培養細胞 vWF陽性表達率為99.0% ± 0.7%，CD31陽性表達率為98.0% ± 1.2%，提示培養細胞為血管內皮細胞。MTT檢測示，隨培養時間延長，血管內皮細胞增殖速度明顯提高。結論采用酶消化法從犬臍靜脈中分離培養血管內皮細胞操作簡便，且細胞數量多、純度高。

引用本文： 胡泉,柴家科,劉玲英,侯玉森,王一賀,李百玲,楊紅明. 犬臍靜脈血管內皮細胞的分離培養與鑒定. 中國修復重建外科雜志, 2013, 27(4): 460-463. doi: 10.7507/1002-1892.20130105 復制

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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.	黃起壬, 戴育成, 李劍, 等. 三種人臍靜脈內皮細胞分離方法的比較. 江西醫學院學報, 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.	張臣, 李彤, 侯躍龍, 等. 人臍靜脈血管內皮細胞的高效分離與培養. 天津醫藥, 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.	劉長樂, 鄭心田, 李廣平, 等. 體外人臍靜脈內皮細胞的培養鑒定及抗原表達分析. 山東醫藥, 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. 胡靜, 仲照東, 鄒萍. 血管內皮細胞的生物學特性. 國際輸血及血液學雜志, 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. 黃起壬, 戴育成, 李劍, 等. 三種人臍靜脈內皮細胞分離方法的比較. 江西醫學院學報, 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. 張臣, 李彤, 侯躍龍, 等. 人臍靜脈血管內皮細胞的高效分離與培養. 天津醫藥, 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. 劉長樂, 鄭心田, 李廣平, 等. 體外人臍靜脈內皮細胞的培養鑒定及抗原表達分析. 山東醫藥, 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.

《中國修復重建外科雜志》

犬臍靜脈血管內皮細胞的分離培養與鑒定

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《中國修復重建外科雜志》

犬臍靜脈血管內皮細胞的分離培養與鑒定

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