Ng Chin Tat, Yip Wai Kien, Mohtarrudin Norhafizah, Seow Heng Fong
Universiti Putra Malaysia, Faculty of Medicine and Health Sciences, Department of Pathology, 43400 UPM Serdang, Selangor, Malaysia.
Malays J Pathol. 2015 Dec;37(3):219-25.
Immortalized human endothelial cells are widely used as in vitro models for debilitating conditions such as cancer, cardiovascular and ocular diseases. Human microvascular endothelial cell (HMEC-1) is immortalized via stable transfection with a gene encoding SV40 large antigen whilst telomerase-immortalized human microvascular endothelial (TIME) cells is immortalized by engineering the human telomerase catalytic protein (hTERT) into primary microvascular endothelial cells. Here, we established a three-dimensional (3D) spheroid invasion assay with HMEC-1 and TIME and compared the difference in their ability to invade through the collagen matrix in response to exogenous growth factors, namely vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF).
TIME and HMEC-1 spheroids were embedded in a collagen matrix. The spheroids were stimulated with exogenous growth factors, namely VEGF (50 ng/mL) and bFGF (200 ng/mL). Twelve points of invasion length from a spheroid was measured using image analysis software, Image J. Three independent experiments were conducted and data was analysis by GraphPad Instat software, version 3.05.
TIME spheroid invasion was 16.5 fold higher with exogenous VEGF (50 ng/mL) and bFGF (200 ng/mL) treatment as compared to those cultured in complete growth medium only. In contrast, no significant difference was observed between HMEC-1 spheroids stimulated with and without exogenous growth factors, VEGF and bFGF.
This is the first report on the establishment of a 3D-spheroid invasion assay with TIME cells. The requirement of VEGF and bFGF for TIME spheroids invasion is a novel finding. In addition, this assay offers an advantage over HMEC-1 for testing novel angiogenic agents since it is not affected by endogenously secreted growth factors.
永生化人内皮细胞被广泛用作体外模型，用于研究诸如癌症、心血管疾病和眼部疾病等使人衰弱的病症。人微血管内皮细胞（HMEC-1）通过用编码SV40大T抗原的基因进行稳定转染而永生化，而端粒酶永生化人微血管内皮（TIME）细胞则通过将人端粒酶催化蛋白（hTERT）导入原代微血管内皮细胞而永生化。在此，我们建立了一种用HMEC-1和TIME细胞进行的三维（3D）球体侵袭试验，并比较了它们在响应外源性生长因子（即血管内皮生长因子（VEGF）和碱性成纤维细胞生长因子（bFGF））时穿过胶原基质的侵袭能力差异。
将TIME和HMEC-1球体包埋在胶原基质中。用外源性生长因子VEGF（50 ng/mL）和bFGF（200 ng/mL）刺激球体。使用图像分析软件Image J测量球体的12个侵袭长度点。进行了三个独立实验，并使用GraphPad Instat软件3.05版进行数据分析。
与仅在完全生长培养基中培养的球体相比，用外源性VEGF（50 ng/mL）和bFGF（20 ng/mL）处理后，TIME球体的侵袭能力提高了16.5倍。相比之下，在有和没有外源性生长因子VEGF和bFGF刺激的HMEC-1球体之间未观察到显著差异。
这是关于用TIME细胞建立3D球体侵袭试验的首次报告。VEGF和bFGF对TIME球体侵袭的需求是一项新发现。此外，该试验在测试新型血管生成剂方面比HMEC-1具有优势，因为它不受内源性分泌生长因子的影响。
