【摘要】 背景:目前关于联合应用Caspase抑制剂及Cocktail蛋白酶抑制剂在胰岛细胞分离纯化过程中对胰岛细胞的影响的研究还较少有报道。目的:比较Caspase抑制剂及Cocktail蛋白酶抑制剂对胰岛细胞在分离纯化及培养过程中的保护作用。方法:取新生猪,体外分离、纯化和培养猪胰岛,培养24 h后分为3组:1空白对照组。2消化时加抑制剂组仅在消化过程中加入Caspase抑制剂及Cocktail蛋白酶抑制剂。3消化及培养时加抑制剂组在消化及培养的过程中均加入Caspase抑制剂及Cocktail蛋白酶抑制剂。AO-EB染色定性观察细胞形态及凋亡情况,流式细胞术定量检测细胞活力及凋亡。结果与结论:空白对照组β细胞所占百分比为66.91%,消化时加抑制剂组为84.58%,消化及培养时加抑制剂组为87.15%;空白对照组活细胞、凋亡细胞及死亡细胞的比例分别为56.52%、16.15%、21.25%,消化时加抑制剂组分别为62.27%、14.66%、14.47%,消化及培养时加抑制剂组分比为73.09%、6.83%、10.28%。结果表明,在消化以及体外培养的过程中均加入Caspase抑制剂及Cocktail蛋白酶抑制剂可明显减少细胞的损失,并且可以增加胰岛β细胞的百分比。更多还原

【Abstract】 BACKGROUND;Currently,there are few reports on the effects of Caspase inhibitors combined with Cocktail protease inhibitors on isolation and purification of islet cells.OBJECTIVE;To explore the effects of Caspase inhibitors and Cocktail protease as protectors on islet cells during isolation and purification process.METHODS;New-born pigs were selected to separate,purify and culture the islet cells,and after 24 hours of culture,the specimens were divided into three groups;blank control group,experimental group A(Caspase inhibitors and Cocktail protease were added only during digestion process),and experimental group B(Caspase inhibitors and Cocktail protease were added both during digestion and culture processes).AO-EB staining was used to qualitatively observe cell morphology and apoptosis.Flow cytometry was employed to quantitatively detect cell viability and apoptosis.RESULTS AND CONCLUSION;Percentages of β cells were 66.91% in blank control group,84.58% in the experimental group A,and 87.15% in the experimental group B.The proportions of living cells,apoptotic cells and dead cells were 56.52%,16.15%,21.25% in the blank control group,62.27%,14.66%,14.47% in the experimentalgroup A,and 73.09%,6.83%,10.28% in the experimental group B,respectively.These findings indicate that in the process of digestion and culture in vitro,Caspase inhibitors and Cocktail protease inhibitors can both obviously decrease the loss of cells,and increase the percentage of islet beta cells.更多还原