Influence of heme oxygenase-1 gene transfer on the viability and function of rat islets in in vitro culture.
OBJECTIVE
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).


CONCLUSIONS
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.
Chen
Xiao-Bo
XB
Department of Renal Transplantation and Urology, the First People's Hospital, Shanghai Jiao Tong University, China.
Li
Yong-Xiang
YX
Jiao
Yang
Y
Dong
Wei-Ping
WP
Li
Ge
G
Chen
Jing
J
Tan
Jian-Ming
JM
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D002470:Cell Survival / Q000502:physiology*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D022761:Gene Transfer, Horizontal / Q000502:physiology*; D005947:Glucose / Q000494:pharmacology; D051547:Heme Oxygenase-1 / Q000235:genetics* / Q000502:physiology; D007328:Insulin / Q000378:metabolism; D007515:Islets of Langerhans / Q000166:cytology / Q000201:enzymology* / Q000502:physiology*; D016381:Islets of Langerhans Transplantation; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection
D007328:Insulin; D051547:Heme Oxygenase-1; D005947:Glucose