大鼠胰岛分离纯化和功能分析的研究
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江苏省卫生厅135工程项目(苏卫科教[2001]34号)


Isolation, purification and functional aanalyses of rat lslets
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    摘要:

    目的:探讨大鼠胰岛分离纯化的方案。方法:采用胰管内灌注胶原酶Ⅴ消化、Histopaque-1077密度梯度离心分离纯化20只雄性SD大鼠胰岛。椎虫蓝染色判断胰岛存活率,双硫腙染色后计数胰岛,葡萄糖刺激胰岛素释放实验评估胰岛功能,同种异体胰岛门静脉移植治疗5只链脲佐菌素诱导的糖尿病大鼠,监测血糖变化。结果:分离纯化后平均胰岛得率为(646 ± 67)个/大鼠,存活率达到90%以上。新鲜分离的胰岛呈椭圆或圆形,悬浮于培养液面,胞膜光滑完整,直径大于150 μm的细胞团占95%以上。葡萄糖刺激的胰岛素释放试验结果表明:第1、3、5天胰岛的刺激指数分别为2.6倍、1.7倍和1.4倍,两组间低糖和高糖的胰岛分泌量差异均有显著性(P < 0.05)。胰岛门静脉移植术后,第2天大鼠血糖降至11.1 mmol/L,8天内血糖可控制在15 mmol/L以下,随后血糖逐步升高。结论:采用胰管内灌注胶原酶Ⅴ消化联合Histopaque-1077分离纯化是一种较好的大鼠胰岛分离方法,胰岛得率较高且功能良好。

    Abstract:

    Objective:To optimize the isolation and purification of rat islets. Methods:Pancreas of SD rats were digested by collagenase V and purified by Histopaque-1077. The islets were stained with trypan blue to identify the viability, and stained with dithezone to count the yield. Glucose stimulated insulin secretion(GSIS) of islets was performed in vitro. Allogeneic islets transplantation via portal vein was performed in STZ-induced diabetic SD rats and the blood glucose level was observed. Results:The average yield was 646 ± 67 perrat and the livability rate was above 90%. GSIS showed that SI(stimulating index) was 2.6, 1.7 and 1.4 at the 1 st, 3 rd, 5 th respectively. There was significant difference between low glucose groups and high glucose groups(P < 0.05). After transplantation, the blood glucose decreased to 11.1 mmol/L at the 2nd day and kept below 15 mmol/L for 8 days. Conclusion:Collagenase V digestion combined with Histopaque-1077 purification is an effective method for rat islets isolation, and the islet yield is high with good function.

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刘翠萍,徐宽枫,武晓泓,茅晓东,刘 超.大鼠胰岛分离纯化和功能分析的研究[J].南京医科大学学报(自然科学版),2007,(3):242-245

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  • 收稿日期:2006-09-05
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