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目的介绍一种密度梯度离心分离大鼠腹腔肥大细胞(RPMC)的技术。方法比较改良方法和原方法2种分离的细胞量、活性及纯度。台盼蓝染色检测细胞存活率;甲苯胺蓝染色检测肥大细胞纯度。将弃去密度梯度离心上液2 m(lA方法)与2.5 m(lB方法)比较,以分析弃去的细胞液的量与肥大细胞纯度的关系。用C48/80刺激分离的RPMC,检测细胞释放的β-氨基己糖苷酶和组胺以确定分离的RPMC脱颗粒活性;透视电镜扫描观察细胞。结果改良方法分离的细胞显著多于原方法分离的细胞数;两种方法分离的细胞存活率都在99%以上;肥大细胞纯度都在90%以上。C48/80刺激RPMC,分离的细胞释放大量的β-氨基己糖苷酶和组胺,高于空白对照组。电镜观察细胞具有明显的肥大细胞形态。结论改良的Percoll密度梯度离心分离大鼠腹腔肥大细胞的方法可行。
Objective To introduce a technique of density gradient centrifugation of rat peritoneal mast cells (RPMC). Methods The cell number, activity and purity of the two methods were compared by the improved method and the original method. Cell viability was detected by trypan blue staining. Mantle cell purity was detected by toluidine blue staining. Discard the density gradient centrifugation of 2 m (lA method) and 2.5 m (lB method) to analyze the amount of discarded cell fluid and mast cell purity. Isolated RPMCs were stimulated with C48 / 80, beta-hexosaminidase released from the cells and histamine were assayed to determine isolated RPMC degranulation activity; cells were observed under a scanning electron microscope. Results The number of cells isolated by the improved method was significantly higher than that of the original method. The survival rates of cells isolated by both methods were above 99%. The purity of mast cells was over 90%. C48 / 80 stimulated RPMC, isolated cells release large amounts of β-hexosaminidase and histamine, higher than the blank control group. Electron microscopy cells have obvious mast cell morphology. Conclusion The modified Percoll density gradient centrifugation of rat peritoneal mast cells feasible.