大鼠胰岛细胞分离纯化的实验研究  被引量:1

Experimental studies on the method of islets isolation and purification in rats

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作  者:庞新路[1] 薛武军[2] 冯新顺[2] 田晓辉[2] 滕琰[2] 郭奇[2] 何晓利[2] 

机构地区:[1]郑州大学第一附属医院器官移植科,郑州450052 [2]西安交通大学医学院第一附属医院

出  处:《陕西医学杂志》2006年第10期1238-1240,1243,共4页Shaanxi Medical Journal

基  金:教育部科学技术重点项目(104169)

摘  要:目的寻找简便、高效的大鼠胰岛分离纯化方法,并对纯化胰岛细胞的基本生物学状况进行鉴定。方法采用胰管内注射胶原酶水浴消化以及Ficoll400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和糖尿病大鼠移植判定胰岛细胞功能。结果纯化后获得(738±193)个胰岛细胞/每只胰腺,纯度为(77±13)%,纯化后细胞形态完好,活度大于95%,体外葡萄糖刺激胰岛素分泌实验,低糖情况下胰岛素分泌量为(24.31±5.47)mIU/L,高糖情况下为(37.62±4.29)mIU/L,两者有显著性差异(P<0.05),胰岛移植后,实验组48h后血糖值降至正常,维持(3.9±1.7)d。结论采用胰管内胶原酶灌注进行大鼠胰岛分离及Ficoll400不连续密度梯度法纯化可获得较高纯度和活度的胰岛细胞。Objective: To develop a simple, effective method of islet isolation and purification in rats , and to assess the biological function of the purified islet in vitro and in vivo. Methods : Collagenase was injected into pancreatic duct followed by incubation in water bath to digest the pancreas and isolate islet, then discontinuous gravity gradient purification was used to purify the islet. The identification of purified islets was evaluated by dithizone staining. The viability of islets was assessed by fluorescence staining of aridine orange (AO) and propidium iodide (PI). The function of purified islets was determined by Glucose-stimulated insulin release test and transplantation of rat with streptozocin-induced diabetes. Results: (738±193) islets were recovered after purification. The average purity was (77±13) %, the viability of islets was more than 95%. When inspected by glucose stimulation, the secreted insulin concentration was (24. 31±5.47) mIU/L when stimulated by low concentration glucose and (37.62±4. 29)mIU/L by high concentrationglucose. There was significant difference between the two phases (P〈0. 05). The concentration of blood sugar recovered to normal level after two days in the group of islet transplantation. Conclusion: More islets can be procured with good function and shape by using the method of injecting collagenase into pancreatic duct followed by incubation in water bath and purification by discontinuous gravity gradient.

关 键 词:胰岛/细胞学 胰岛移植 大鼠 

分 类 号:R587.1[医药卫生—内分泌]

 

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