不同分离及纯化方法对人胰岛分离纯化的影响

Effect of different methods of purification and separation on human islet isolation purification

目的:探讨胶原酶P、释放酶分离及不连续密度梯度和连续密度梯度纯化方法对胰岛产量、纯度及活动度的影响。方法:分别采用胰管内注射胶原酶P及释放酶水浴消化以及分别采用COBE2991连续密度梯度离心法和Ficoll不连续密度梯度离心法纯化。用DTZ染色计算胰岛细胞的纯度;用AO/PI染色计算胰岛细胞存活率;通过体外葡萄糖刺激胰岛素分泌试验判定胰岛细胞功能。结果:两组活度均在95%左右,两组之间差异无统计学意义(P>0.05)。应用Ficoll不连续密度梯度离心法可获得胰岛纯度(77.5±13.4)%,用胶原酶P组,释放酶组可分别获得(1 829±517)和(3129±893)个胰岛/每克胰腺,两者差异有统计学意义(P<0.05)。应用COBE2991连续密度梯度离心法可获得胰岛纯度(92.8±7.4)%,纯化后细胞形态完好,两者差异有统计学意义(P<0.05)。体外葡萄糖刺激胰岛素分泌实验,高糖情况下(5.33±1.13)m IU/L,两者差异有统计学意义(P<0.05),低糖情况下胰岛素分泌量(2.63±0.67)m IU/L。结论:用COBE2991连续密度梯度离心法纯化胰岛较Ficoll400不连续密度梯度法纯化可获得纯度更高的胰岛细胞。采用胰管内释放酶灌注进行人胰岛分离优于胶原酶P。

Objective: To investigate the effect of release and enzyme isolated by collagenase P and discontinuous density gradient and continuous density gradient purification method on the islet yield,purityand activity.Methods:We respectively used ductal injection of collagenase P and release of enzyme water bath digestion and respectively used COBE2991 continuous density gradient centrifugation and discontinuous Ficoll density gradient centrifugation method to purified.We used the DTZ staining to calculate the purity of islet cells;we used AO/PI staining to calculate islet cell survival rate;through the vitro glucose stimulated insulin secretion test to determine the islet cell function.Results:The activity of the two groups were at about 95%,and there were no statistically significant difference between the two groups(P>0.05).In the collagenase P group,the release enzyme group could be obtained separately(1829±517) and(3129±893) islets per gram of pancreatic,both of which had significant difference(P<0.05).We used the ficoll discontinuous density gradient centrifugation can obtain islet purity(77.5±13.4)%;we used COBE2991 continuous density gradient centrifugation can obtain islet purity(92.8 ± 7.4)%;the cell morphology were intact after the purification,and both of them had significant difference(P<0.05).Glucose stimulated insulin secretion experiments,under the condition of low sugar insulin secretion(2.63 ± 0.67)u U/islet, high glucose condition(5.33 ± 1.13) u U/islet, both of which had significant difference(P<0.05).Conclusion:The COBE2991 continuous density gradient centrifugation method to purified islets can get higher purity islet cells than ficoll400 discontinuous density gradient method.The pancreatic duct releasing enzyme perfusion to isolate human islet is better than collagenase P.