大鼠胰岛分离纯化新方法“两步过筛法”的建立及评估

The establishment of “two-step sequential filtration method” on the yield rate of purified islets in rats

摘要:

目的：建立胆管内灌注胶原酶分离胰岛与适宜孔径滤网过筛相结合的快速纯化胰岛细胞的方法并评估这种方法的效果。方法：清洁级8～12周龄SpragueDawley大鼠胆总管内灌注胶原酶消化、分离胰岛，分别采用Ficoll不连续密度梯度离心法和两次经不同孔径滤网过滤的两步过筛法纯化胰岛细胞，行双硫腙（dithizone，DTZ）染色、吖啶橙/碘丙啶(acridine orange / propidium iodide, AO/PI)双色荧光染色法分析分离胰岛的纯度与活率；行葡萄糖刺激—胰岛素释放试验检测细胞活性；采用免疫组化荧光染色法观察胰岛细胞胰岛素的合成功能。结果：大鼠两步过筛法和胰岛密度梯度离心法胰岛细胞收获量分别782±115 胰岛当量（IEQ）和598±135 IEQ，差异有统计学意义（P<0.01）；纯度分别为90％～100％和65％～85%，活率分别为>95％和85%～95%。两步过筛法分离的胰岛葡萄糖刺激—胰岛素释放试验显示培养24 h后，高糖实验组胰岛素浓度（76.9±6.1 μg/L）显著高于刚分离纯化后即加入高糖刺激时的浓度（49.4±3.9 μg/L），差异有统计学意义（P<0.01）。结论：两步过筛法分离纯化大鼠胰岛可获得纯度高、活率高且活性好的大鼠胰岛细胞。

Abstract:

OBJECTIVE: To develop a simple, rapid and reliable method of purifying SpragueDawley (SD) rat islets by sequential filtration through two cell strainers of different sizes and to evaluate the efficacy of the method. METHODS: Islets were isolated from 8 to 12-week-old clean grade Sprague-Dawley rat pancreases using the standard collagenase digestion procedure and purified with either the generally used Ficoll density gradient method or the innovative two-step sequential filtration method. The purity and vitality of the isolated islets were visualized and assessed with DTZ and AO/PI staining. Glucose stimulating tests were performed to assay cell activity, and immunohistochemical staining was used to evaluate the synthesis function of islet cells. RESULTS: The yield of islets in the two-step filtration method group was 782±115 IEQ per rat, which was significantly higher than in the conventional Ficoll density gradient method group (598±135 IEQ per rat, P<0.01). Purity of the isolated islets in the two-step filtration method group was 90%-100% and vitality was over 95%. In the conventional Ficoll density gradient method group, islet purity was 65%-85% and vitality was 85%-95%. With regard to the high-sugar stimulation test in the two-step filtration method group, insulin concentrations in islets cultured for 24 hours were significantly higher than in those that were freshly purified (76.9 ± 6.1 μg/L vs 49.4 ± 3.9 μg/L; P<0.01). CONCLUSIONS: A two-step sequential filtration method for rat islet purification was developed and the method was simple and reliable, with high islet vitality, purity and yield.