小鼠胰岛密度梯度离心方法的研究  

Method of gradient centrifugation for mouse pancreatic islet isolation

作　　者：张金花[1] 邵益[1] 厉智威[1] 方程[1] 田阳[1] 严盛[1] 郑树森[1] 

机构地区：[1]浙江大学医学院附属第一医院卫生部多器官联合移植重点实验室,杭州310003

出　　处：《浙江医学》2013年第24期2152-2154,共3页Zhejiang Medical Journal

基　　金：浙江省科技厅创新团队项目(2009R50038)

摘　　要：目的探索最优的密度梯度离心方案,提高胰岛的纯度和得率,为胰岛移植相关研究奠定基础。方法采用雄性Balb/c小鼠作为供体分离胰岛,以0.5mg/ml胶原酶XI灌注和消化胰腺,通过不同的密度梯度离心策略分离纯化胰岛,进行双硫腙(DTZ)特异染色法染色,显微镜下观察胰岛的纯度和得率,并通过吖啶橙(AO)/碘化丙啶(PI)染色检查分离获得的胰岛活性,最终确定最佳的密度梯度离心方法。结果Balb/c小鼠在Ficoll-1 108(4ml)、1.096(2ml)、1.069(2ml)、HBSS(2ml),加速度1、减速度1、400r/min、4min中转1 800r/min、8min离心后胰岛得率为(125±39)IEQ、纯度为(79.6±10.5)%,经相同条件二次梯度后得率无明显降低但纯度明显提高,达(96.2±1.4)%。经AO/PI染色鉴定分离胰岛活性良好。结论本研究所采用的小鼠胰岛密度梯度离心方法很有效,能稳定地获得纯度较高且数量可观的胰岛。Objective To establish a stable method for mouse pancreatic islet isolation. Methods Male Balb/c mice were used for islet harvest and isolation. The pancreas were digested by 0.5mg/ml collagenase, and the islets were isolated and puri- fied with different gradient centrifugation strategy, stained with DTZ, and observed by microscopy to determine an optimal proto- col for islet isolation. AO/PI staining was used to detect the activity of isolated islets. Different protocols were compared using X2 and one-way analysis of variance （ANOVA）. Results The optimal conditions used for isolation of pancreatic islets from Balb/c mice were as follows： using Ficoll -1.108 （4ml）, 1.096 （2ml）, 1.069 （2ml）, HBSS （2ml）, acceleration 1-deceleration 1,400rpm for 4min transfer to 1800rpm for another 8min. The preliminary yield was （125±39）IEQ, purity was （79.6±10.5）%, then underwent another gradient centrifugation with the same strategy, the purity significantly increased to （96.2±1.4）%, while the yield was still satisfying. AO/PI staining confirmed good activity of the isolated islets. Conclusion The modified islet isolation method is a better choice to obtain higher purity and considerable numbers of islets.

关 键 词：胰岛 分离纯化 密度梯度离心 

分 类 号：R587.1[医药卫生—内分泌]