使用HUVEC細胞進行血管生成實驗，并用LONZA核轉儀轉染anti-VEGFR2 siRNA
Srinivasan Kokatam1, Kanchan Tiwari1, Jenny Schroeder2, Andrea Toell2, Lubna Hussain3, Preeti Kapoor1.
1. Lonza India Pvt Ltd, Hyderabad, India; 2. Lonza Cologne GmbH, Cologne, Germany; 3. Lonza Walkersville, Inc., Walkersville, MD, U.S.A.
實驗材料：
英文名 | 中文名 | 品牌 | 貨號 |
single donor Clonetics? HUVECs | 臍靜脈內皮細胞，單一供體 | LONZA Clonetics | C2517A |
EGM? 2 Media | 血管內皮細胞培養基 | LONZA | CC-3162 |
ViaLight? Plus BioAssay Kit | 細胞活力檢測試劑盒 | LONZA | LT07-221 |
Matrigel | 基質膠 | Corning | 356237 |
4D Nucleofector | 核轉儀 | LONZA | C+X+96模塊 |
P5 Primary Cell Nucleofector Kit | 核轉試劑盒 | LONZA | V4XP-5024 V4XP-5032 |
Calcein AM | Life Technologies | C3100MP | |
VEGFR2 ELISA | VEGFR2的ELISA檢測試劑盒 | R&D Systems | DYC1780-5 |
培養基配方：
實驗流程簡介：
1. 使用pmaxGFP質粒進行進行預實驗，優化HUVEC細胞核轉條件；
2. 檢測核轉條件對HUVEC細胞的Tube formation是否有影響；
3. 轉染siRNA，確定對細胞活力影響最小的最佳濃度，確定檢測蛋白表達降低的最佳時間點；
4. 評價siRNA對HUVEC細胞Tube Formation的影響。
實驗結果：
圖1. HUVEC細胞轉染GFP質粒，轉染效率約為82%，對細胞形態無明顯影響：
圖2. HUVEC細胞進行核轉后，對Tube formation無明顯影響：
圖3. Anti-VEGFR2 siRNA轉染后的細胞對VEGF無反應，證明siRNA發揮作用。
參考文獻：
1. Timar, J. et. al. (2001) Angiogenesis-dependent diseases and angiogenesis therapy. Pathol. Oncol. Res.7 (2): 85-94
2. Murga M1, Fernandez-Capetillo O, Tosato G. (2005) Neuropilin-1 regulates attachment in human endothelial cells independently of vascular endothelial growth factor receptor-2. Blood 105 (5): 1992-9
3. Yang, S, Xin, X, Zlot, C et. al. (2001) Vascular Endothelial Cell Growth Factor-Driven Endothelial Tube Formation Is Mediated by Vascular Endothelial Cell Growth Factor Receptor-2, a Kinase Insert Domain-Containing Receptor. Arterioscler. Thromb. Vasc. Biol. 21: 1934-1940
4. Zumbansen M, Altrogge L, Toell A, Leake D, Muller-Hartmann H (2009) First siRNA Library Screening in Difficult-to-Transfect HUVEC and Jurkat Cells. White paper.
5. Zeitelhofer M, Vessey JP, Xie Y, Tubing F, Thomas S, Kiebler M, Dahm R (2007) High-efficiency transfection of mammalian neurons via Nucleofection. Nat. Protocols 2: 1692–1704
6. Kapoor, R., Reddy, V. and Kapoor, P. (2014) Tube Formation Assay with Primary Human Umbilical Vein Endothelial Cells. Resource Notes, 2014: 9-12.