摘要
目的建立一种经济高效的大鼠胰岛细胞分离纯化方法,为胰腺的修复重建奠定实验基础。方法成年雄性SD大鼠25只,体重230～380g,共进行5次实验,每5只大鼠一组进行消化和分离。采用医用复方氯化钠注射液(compound sodium chloride injection,CSCI)经胰总管灌注大鼠胰腺,0.5mg/mLⅤ型胶原酶消化后,分别采用浓度为27.0%、23.0%、20.5%和11.0%的Ficoll 400形成不连续密度梯度介质,离心纯化胰岛细胞。双硫腙(dithizon,DTZ)染色行纯化前后胰岛细胞计数和纯度检测;荧光染料碘化丙啶(propidium iodide,PI)和二乙酸荧光素(fluorescein diacetate,FDA)储存液双染色鉴定胰岛细胞活性;RPMI1640培养基培养3d后,分别用浓度为2.8mmol/L的低糖和25.0mmol/L的高糖行葡萄糖刺激胰岛素释放实验检测胰岛细胞功能。结果5次实验胰岛细胞消化时间为(13.8±1.6)min。DTZ染色鉴定纯化前胰岛细胞数为(5626±422)个,纯化后为(2914±485)个,纯化后的胰岛细胞数较纯化前明显减少(P<0.01),回收率51.6%±6.0%,每个胰腺收获胰岛细胞数为(583±97)个/只。5次分离获得的胰岛细胞纯度为90.2%±3.4%,活性为81.6%±7.0%。培养3d后,葡萄糖刺激胰岛素释放实验显示:低糖环境下胰岛素水平为(39.7±7.5)EU/L,高糖环境为(116.1±17.4)EU/L,比较差异有统计学意义(P<0.01);刺激指数为3.0±0.4。结论采用CSCI作为大鼠胰岛细胞分离纯化的主要液体试剂,并采用低浓度Ⅴ型胶原酶消化,不仅可降低实验成本,同时可获得高质量的胰岛细胞。
Objective To set up an economic and effective method for islet isolation from rat, and thereby prove a laboratory protocol of animal model for clinical islet transplantation. Methods Twenty-five adult male SD rats weighing 230-380 g were used as organ donor. In each of 5 repeated experiments, pancreatic islets of 5 animals were isolated by intraductal infusion of compound sodium chloride injection （CSCI）, and subsequently, digested with low concentration （0.5 mg/mL） of collagenase V solution. Islet purification was performed by using a discontinuous density gradient centrifugation that was prepared with 27.0%, 23.0%, 20.5% and 11.0% of Ficoll 400. Islet yield and purity were determined by dithizon （DTZ） stain, and propidium iodide （PI）/fluorescein diacetate （FDA） double stain was used to check viability of islets. The endocrine secretory function was assessed by insulin secretion in either low （2.8 mmol/L） or high （25.0 mmol/L） glucose incubation after 3 days of culture in RPMI1640 media. Results Average islet digestion time of 5 experiments was （13.8 ± 1.6） min. Before purification, average isolated number was （5 626 ± 422） islets, and the number was significantly reduced to （2 914 ± 485） islets after purification （P 〈 0.01）. The average recovery rate was 51.6%± 6.0%, and the average yield was （583±97） islets/pancreas. The average purity and viability of islets were 90.2% ± 3.4% and 81.6% ± 7.0%, respectively. After 3 days of culture, insulin secretion of the islets was （116.1±17.4） EU/L in high glucose incubation, which was significantly higher than that of low glucose environment [（39.7 ± 7.5） EU/L, P 〈 0.01）]. The average insulin stimulation index was 3.0 ± 0.4. Conclusion The islet isolation with the CSCI solution and digestion with low concentration of collagenase V decrease experimental cost and also have a beneficial effect on islet recovery and their function.
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2008年第5期610-613,共4页
Chinese Journal of Reparative and Reconstructive Surgery
基金
教育部博士点基金资助项目(20050610094)~~
关键词
大鼠胰岛细胞
分离纯化
细胞培养
Rat islet cell Isolation and purification Cell culture
