Basic Research
Copyright ©2007 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Gastroenterol. Feb 21, 2007; 13(7): 1053-1059
Published online Feb 21, 2007. doi: 10.3748/wjg.v13.i7.1053
Influence of heme oxygenase-1 gene transfer on the viability and function of rat islets in in vitro culture
Xiao-Bo Chen, Yong-Xiang Li, Yang Jiao, Wei-Ping Dong, Ge Li, Jing Chen, Jian-Ming Tan
Xiao-Bo Chen, Yong-Xiang Li, Yang Jiao, Wei-Ping Dong, Jian-Ming Tan, Department of Renal Transplantation and Urology, the First People's Hospital, Shanghai Jiao Tong University; Shanghai Clinical Medical Center of Organ Transplantation , Shanghai 200080, China
Ge Li, Jing Chen, State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200 433, China
Author contributions: All authors contributed equally to the work.
Supported by the National Natural Science Foundation of China, No. 30571759 and Social Development Foundation of Shanghai, No. 2002-53
Correspondence to: Dr Jian-Ming Tan, Department of Renal Transplantation and Urology, the First People's Hospital, Shanghai Jiao Tong University; Shanghai Clinical Medical Center of Organ Transplantation, Shanghai 200080, China. jmtan156@yahoo.com.cn
Telephone: +86-21-63069482 Fax: +86-21-63069482
Received: October 14, 2006
Revised: November 2, 2006
Accepted: November 14, 2006
Published online: February 21, 2007
Abstract

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.

Keywords: Islet viability; Islet function; Heme oxygenase-1; Gene transfer; Adenoviral vectors