使用HUVEC细胞进行血管生成实验，并用LONZA核转仪转染anti-VEGFR2 siRNA，评估其抗血管生成的效果

发布时间：2018年08月03日

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.

实验材料：

英文名	中文名	品牌	货号
single donor Clonetics™ HUVECs	脐静脉内皮细胞，单一供体	LONZA Clonetics	C2517A
EGM™ 2 Media	血管内皮细胞培养基	LONZA	CC-3162
ViaLight™ Plus BioAssay Kit	细胞活力检测试剂盒	LONZA	LT07-221
Matrigel	基质胶	Corning	356237
4D Nucleofector	核转仪	LONZA	C+X+96模块
P5 Primary Cell Nucleofector Kit	核转试剂盒	LONZA	V4XP-5024 V4XP-5032
Calcein AM		Life Technologies	C3100MP
VEGFR2 ELISA	VEGFR2的ELISA检测试剂盒	R&D Systems	DYC1780-5

 

培养基配方：

 

实验流程简介：

1. 使用pmaxGFP质粒进行进行预实验，优化HUVEC细胞核转条件；

2. 检测核转条件对HUVEC细胞的Tube formation是否有影响；

3. 转染siRNA，确定对细胞活力影响最小的最佳浓度，确定检测蛋白表达降低的最佳时间点；

4. 评价siRNA对HUVEC细胞Tube Formation的影响。

 

实验结果：

图1. HUVEC细胞转染GFP质粒，转染效率约为82%，对细胞形态无明显影响：

图2. HUVEC细胞进行核转后，对Tube formation无明显影响：

图3. Anti-VEGFR2 siRNA转染后的细胞对VEGF无反应，证明siRNA发挥作用。

 

参考文献：

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.