新生猪胰岛细胞团的体外培养体系

In Vitro culture system of porcine neonatal pancreatic cell clusters

目的分离纯化新生猪胰岛细胞团(NPCCs),以不同的体外培养方式促进NPCCs成熟,筛选最佳方案。方法利用供龄分别为3天的新生猪,参考Bonner-Weir法和Hill法分离纯化NPCCs,分别以培养体系Ⅰ(90%DMEM、10%胎牛血清、双抗、2 mmol/L谷氨酰胺+0.01%大豆胰酶抑制剂)和培养体系Ⅱ(90%DMEM、10%胎牛血清、双抗、2 mmol/L谷氨酰胺+0.01%大豆胰酶抑制剂、10 mmol/L烟碱、10 ng/mL表皮细胞生长因子、15 ng/mL胰岛素样生长因子)体外培养14天,进行抗胰岛素、抗CK7双荧光染色和胰岛素刺激释放实验。结果应用培养体系Ⅰ的NPCCs体外培养14天时,抗胰岛素阳性细胞(β细胞)百分比为(28.6±5.3)%,显著低于培养体系Ⅱ的(62.1±9.0)%(P(0.01);培养体系Ⅰ的抗CK7阳性细胞(原始导管细胞)百分比为(52.8±7.2)%,显著高于培养体系Ⅱ的(21.5±6.9)%(P(0.01)。培养体系Ⅰ的NPCCs体外培养14天时胰岛素刺激释放量为:低糖:(14.3±4.8)mU/L,高糖:(23.9±6.2)mU/L,高糖+茶碱:(38.5±8.2)mU/L,显著低于培养体系Ⅱ的释放量:(26.2±5.1)mU/L、(46.3±7.0)mU/L、(81.5±10.4)mU/L(P(0.01)。结论培养体系Ⅱ较培养体系Ⅰ更能促进NPCCs中β细胞发育成熟及CK7阳性细胞向β细胞转化,并使胰岛素刺激释放量提高。培养体系Ⅱ是微囊和移植前体外培养NPCCs的理想方案之一。

Objective Isolation,purification and different methods of culture in vitro were approached for selection of preferred culture program of neonodal pancreatic cell clusters. Methods According to methods recorded by Bonner-Weir and Hill,3-day newborn pigs were adopted in isolation and purification of NPCCs which were in turn incubated for 14 days in two culture systems,namely System I （90% DMEM, 10% fetal calf serum,double antibodies,2 mmol/L glutamine ＋ 0.01% soybean trypsin inhibitor） and system Ⅱ （90% DMEM, 10% fetal calf serum, double antibodies,2 mmol /L glutamine ＋ 0.01% soybean trypsin inhibitor, 10 mmol/L NIC, 10 ng/mL EGF, 15 ng/mL IGF -1 ） , respectively, and then treated by anti-insulin and anti-CK7 double fluorescent staining and insulin release test. Results Fifteen days after culture in vitro, ratio of positive anti-insulin cells（ β cell） was significantly lower in System Ⅰ Group than in System Ⅱ Group （28.6% ±5.3% vs72.1% ±9.0%;P〈0.01）;Ratio of positive anti-CK7 cells （primitive duct cell） was higher in System Ⅰ group than in System Ⅱ group （52.8% ±7.2% vs. 21.5%± 6.9% ;P 〈 0.01 ）. Insulin release amount was examined on the 14th day after culture in vitro by low level of sugar as （ 14.3 ± 4.8 ） mU/L, high level of sugar as （23.9 ± 6.2） mU/L, high level sugar ＋ theocin as （38.5 ± 8.2）mU/L, significantly smaller in System I Group than in System Ⅱ Group （26.2 mU/L ± 5. 1 mU/L,46.3 mU/L ± 7.0 mU/L,81.5 mU/L ± 10.4 mU/L） , respectively （P 〈0.01）. Conclusion Compared with culture system Ⅰ ,the system Ⅱ exert more effects on promotion of cells maturation in NPCCs,transformation of CK 7 positive cells to β cells and improvement of insulin stimulation release amount. Adoption of culture system Ⅱ is an ideal proposal of NPCCs culture in vitro for microencapsule and pretransplant management.