血红素加氧酶-1基因转移对体外培养大鼠胰岛活力及功能的影响。
Influence of heme oxygenase-1 gene transfer on the viability and function of rat islets in in vitro culture.
作者信息
Chen Xiao-Bo, Li Yong-Xiang, Jiao Yang, Dong Wei-Ping, Li Ge, Chen Jing, Tan Jian-Ming
机构信息
Department of Renal Transplantation and Urology, the First People's Hospital, Shanghai Jiao Tong University, China.
出版信息
World J Gastroenterol. 2007 Feb 21;13(7):1053-9. doi: 10.3748/wjg.v13.i7.1053.
AIM
To investigate the influence of heme oxygenase-1 (HO-1) gene transfer on the viability and function of cultured rat islets in vitro.
METHODS
Islets were isolated from the pancreata of Sprague-Dawley rats by intraductal collagenase digestion, and purified by discontinuous Ficoll density gradient centrifugation. Purified rat islets were transfected with adenoviral vectors containing human HO-1 gene (Ad-HO-1) or enhanced green fluorescent protein gene (Ad-EGFP), and then cultured for seven days. Transfection was confirmed by fluorescence microscopy and Western blot. Islet viability was evaluated by acridine orange/propidium iodide fluorescent staining. Glucose-stimulated insulin release was detected using insulin radioimmunoassay kits and was used to assess the function of islets. Stimulation index (SI) was calculated by dividing the insulin release upon high glucose stimulation by the insulin release upon low glucose stimulation.
RESULTS
After seven days culture, the viability of cultured rat islets decreased significantly (92% +/- 6% vs 52% +/- 13%, P < 0.05), and glucose-stimulated insulin release also decreased significantly (6.47 +/- 0.55 mIU/L/30IEQ vs 4.57 +/- 0.40 mIU/L/30IEQ, 14.93 +/- 1.17 mIU/L/30IEQ vs 9.63 +/- 0.71 mIU/L/30IEQ, P < 0.05). Transfection of rat islets with adenoviral vectors at an MOI of 20 was efficient, and did not impair islet function. At 7 d post-transfection, the viability of Ad-HO-1 transfected islets was higher than that of control islets (71% +/- 15% vs 52% +/- 13%, P < 0.05). There was no significant difference in insulin release upon low glucose stimulation (2.8 mmol/L) among Ad-HO-1 transfected group, Ad-EGFP transfected group, and control group (P > 0.05), while when stimulated by high glucose (16.7 mmol/L) solution, insulin release in Ad-HO-1 transfected group was significantly higher than that in Ad-EGFP transfected group and control group, respectively (12.50 +/- 2.17 mIU/L/30IEQ vs 8.87 +/- 0.65 mIU/L/30IEQ; 12.50 +/- 2.17 mIU/L/30IEQ vs 9.63 +/- 0.71 mIU/L/30IEQ, P < 0.05). The SI of Ad-HO-1 transfected group was also significantly higher than that of Ad-EGFP transfected group and control group, respectively (2.21 +/- 0.02 vs 2.08 +/- 0.05; 2.21 +/- 0.02 vs 2.11 +/- 0.03, P < 0.05).
CONCLUSION
The viability and function of rat islets decrease over time in in vitro culture, and heme oxygenase-1 gene transfer could improve the viability and function of cultured rat islets.
目的
研究血红素加氧酶-1（HO-1）基因转移对体外培养的大鼠胰岛活力及功能的影响。
方法
通过导管内胶原酶消化法从Sprague-Dawley大鼠胰腺中分离胰岛，并经不连续Ficoll密度梯度离心法纯化。将纯化的大鼠胰岛用含人HO-1基因的腺病毒载体（Ad-HO-1）或增强型绿色荧光蛋白基因（Ad-EGFP）转染，然后培养7天。通过荧光显微镜和蛋白质免疫印迹法确认转染情况。采用吖啶橙/碘化丙啶荧光染色评估胰岛活力。使用胰岛素放射免疫分析试剂盒检测葡萄糖刺激的胰岛素释放，并用于评估胰岛功能。刺激指数（SI）通过高糖刺激时的胰岛素释放量除以低糖刺激时的胰岛素释放量来计算。
结果
培养7天后，培养的大鼠胰岛活力显著降低（92%±6%对52%±13%，P<0.05），葡萄糖刺激的胰岛素释放也显著降低（6.47±0.55 mIU/L/30IEQ对4.57±0.40 mIU/L/30IEQ，14.93±1.17 mIU/L/30IEQ对9.63±0.71 mIU/L/30IEQ，P<0.05）。以20的感染复数用腺病毒载体转染大鼠胰岛效率高，且不损害胰岛功能。转染后7天，Ad-HO-1转染的胰岛活力高于对照胰岛（71%±15%对52%±13%，P<0.05）。在低糖刺激（2.8 mmol/L）时，Ad-HO-1转染组、Ad-EGFP转染组和对照组之间的胰岛素释放无显著差异（P>0.05），而在高糖（16.7 mmol/L）溶液刺激时，Ad-HO-1转染组的胰岛素释放分别显著高于Ad-EGFP转染组和对照组（12.50±2.17 mIU/L/30IEQ对8.87±0.65 mIU/L/30IEQ；12.50±2.17 mIU/L/30IEQ对9.63±0.71 mIU/L/30IEQ，P<0.05）。Ad-HO-1转染组的SI也分别显著高于Ad-EGFP转染组和对照组（2.21±0.02对2.08±0.05；2.21±0.02对2.11±0.03，P<0.05）。
结论
大鼠胰岛在体外培养过程中活力和功能随时间降低，血红素加氧酶-1基因转移可改善培养的大鼠胰岛的活力和功能。
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