CAJ | 학술논문

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(1829±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)mIU/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)mIU/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。
목적：탐토효원매P、석방매분리급불련속밀도제도화련속밀도제도순화방법대이도산량、순도급활동도적영향。방법：분별채용이관내주사효원매P급석방매수욕소화이급분별채용COBE2991련속밀도제도리심법화Ficoll불련속밀도제도리심법순화。용DTZ염색계산이도세포적순도；용AO/PI염색계산이도세포존활솔；통과체외포도당자격이도소분비시험판정이도세포공능。결과：량조활도균재95%좌우，량조지간차이무통계학의의(P＞0.05)。응용Ficoll불련속밀도제도리심법가획득이도순도(77.5±13.4)%，용효원매P조，석방매조가분별획득(1829±517)화(3129±893)개이도/매극이선，량자차이유통계학의의(P＜0.05)。응용COBE2991련속밀도제도리심법가획득이도순도(92.8±7.4)%，순화후세포형태완호，량자차이유통계학의의(P＜0.05)。체외포도당자격이도소분비실험，고당정황하(5.33±1.13)mIU/L，량자차이유통계학의의(P＜0.05)，저당정황하이도소분비량(2.63±0.67)mIU/L。결론：용COBE2991련속밀도제도리심법순화이도교Ficoll400불련속밀도제도법순화가획득순도경고적이도세포。채용이관내석방매관주진행인이도분리우우효원매P。
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)uU/islet, high glucose condition(5.33 ± 1.13) uU/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.