Hadjivassiliou V, Green M H, Green I C
Biochemistry Department, University of Sussex, Brighton, UK.
Diabetologia. 2000 Sep;43(9):1170-7. doi: 10.1007/s001250051509.
AIMS/HYPOTHESIS: The aim of this study was to develop immunomagnetic purification by the Dynabead system to separate insulin-containing beta cells from a mixed rat islet cell population. Functional studies on insulin secretion and a test of the susceptibility of Dynabead-separated beta cells to DNA damage following cytokine exposure were carried out.
Dynabeads are uniform, paramagnetic particles coated with specific antibodies. Single rat islet cells were initially incubated with the beta-cell surface specific antibody (K14D10 mouse IgG) for 20-60 min. A suspension of Dynabeads coated with a secondary antibody (anti-mouse IgG) was added for a further 15 min, after which the Dynabead-coated cells were instantaneously pelleted by contact between the tube and a magnet (Dynal MPC). Immunocytochemistry was used to confirm that the Dynabead-coated cells contained insulin and to quantify the efficiency of the method. Dynabead-coated and non-coated cells were stained for insulin and glucagon.
Dynabead immunopurification yielded 95% pure insulin-containing beta cells, which released insulin in response to isobutylmethylxanthine and glucagon-like polypeptide 1. The insulin content of Dynabead-coated beta cells was significantly higher than that of non-coated cells. Successful separation was achieved using as few as 30 islets as starting material. Using the comet assay, we found that Dynabead-coated beta cells showed equal susceptibility to cytokine-induced DNA damage as non-coated cells.
CONCLUSION/INTERPRETATION: We conclude that Dynabead separation of beta cells is simple, rapid, applicable to large or small numbers of islets and can be used to study beta-cell specific function and responsiveness.
目的/假设：本研究的目的是开发一种利用Dynabead系统进行免疫磁珠纯化的方法，以从混合的大鼠胰岛细胞群体中分离出含胰岛素的β细胞。对胰岛素分泌进行了功能研究，并检测了细胞因子暴露后Dynabead分离的β细胞对DNA损伤的敏感性。
Dynabeads是包被有特异性抗体的均匀顺磁性颗粒。首先将单个大鼠胰岛细胞与β细胞表面特异性抗体（K14D10小鼠IgG）孵育20 - 60分钟。加入包被有二抗（抗小鼠IgG）的Dynabeads悬浮液，再孵育15分钟，之后通过将试管与磁体（Dynal MPC）接触，使包被有Dynabeads的细胞立即沉淀。采用免疫细胞化学方法确认包被有Dynabeads的细胞含有胰岛素，并对该方法的效率进行定量。对包被和未包被Dynabeads的细胞进行胰岛素和胰高血糖素染色。
Dynabead免疫纯化得到了纯度为95%的含胰岛素β细胞，这些细胞对异丁基甲基黄嘌呤和胰高血糖素样肽1有胰岛素释放反应。包被有Dynabeads的β细胞的胰岛素含量显著高于未包被的细胞。以少至30个胰岛作为起始材料即可成功实现分离。通过彗星试验，我们发现包被有Dynabeads的β细胞与未包被的细胞对细胞因子诱导的DNA损伤具有相同的敏感性。
结论/解读：我们得出结论，Dynabeads分离β细胞的方法简单、快速，适用于大量或少量胰岛，可用于研究β细胞的特定功能和反应性。
