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.

 

 

 

1. 使用pmaxGFP質(zhì)粒進(jìn)行進(jìn)行預(yù)實(shí)驗(yàn)，優(yōu)化HUVEC細(xì)胞核轉(zhuǎn)條件；

2. 檢測(cè)核轉(zhuǎn)條件對(duì)HUVEC細(xì)胞的Tube formation是否有影響；

3. 轉(zhuǎn)染siRNA，確定對(duì)細(xì)胞活力影響最小的最佳濃度，確定檢測(cè)蛋白表達(dá)降低的最佳時(shí)間點(diǎn)；

4. 評(píng)價(jià)siRNA對(duì)HUVEC細(xì)胞Tube Formation的影響。

 

圖1. HUVEC細(xì)胞轉(zhuǎn)染GFP質(zhì)粒，轉(zhuǎn)染效率約為82%，對(duì)細(xì)胞形態(tài)無(wú)明顯影響：

圖2. HUVEC細(xì)胞進(jìn)行核轉(zhuǎn)后，對(duì)Tube formation無(wú)明顯影響：

圖3. Anti-VEGFR2 siRNA轉(zhuǎn)染后的細(xì)胞對(duì)VEGF無(wú)反應(yīng)，證明siRNA發(fā)揮作用。

 

 

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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.