Welch Johanna, Svensson Katrin, Kucharzewska Paulina, Belting Mattias
Department of Clinical Sciences, Section of Oncology, Lund University and Lund University Hospital, Lund, Sweden.
Methods Mol Biol. 2011;720:327-38. doi: 10.1007/978-1-61779-034-8_20.
The polyamines are polycationic compounds essential for cellular proliferation and transformation. In addition to a well-defined biosynthesis pathway, polyamines are internalized into cells by as yet incompletely defined mechanisms. Numerous reports have shown that efficient polyamine uptake depends on the presence of polyanionic, cell surface-associated heparan sulfate proteoglycans (HSPGs). In this chapter, we provide protocols for studying HSPG-mediated uptake of polyamines in various cell lines, and provide instructions for the use of two different genetic models of HSPG deficiency. We describe the enzymatic reduction of cell surface HSPG through Heparinase III lyase treatment as well as the use of phage display-derived single chain variable fragment (scFv) anti-HS antibodies to block HSPGs at the cell surface. Finally, we provide a protocol for the quantitative verification of loss or reduction of cell surface HSPGs and a detailed description of polyamine uptake measurement.
多胺是细胞增殖和转化所必需的聚阳离子化合物。除了明确的生物合成途径外，多胺还通过尚未完全明确的机制内化进入细胞。大量报告表明，高效的多胺摄取依赖于细胞表面相关的多阴离子硫酸乙酰肝素蛋白聚糖（HSPG）的存在。在本章中，我们提供了研究HSPG介导的多胺在各种细胞系中摄取的方案，并提供了使用两种不同的HSPG缺陷遗传模型的说明。我们描述了通过肝素酶III裂解酶处理对细胞表面HSPG进行酶促还原，以及使用噬菌体展示衍生的单链可变片段（scFv）抗HS抗体来阻断细胞表面的HSPG。最后，我们提供了细胞表面HSPG丢失或减少的定量验证方案以及多胺摄取测量的详细描述。
