Effect of mini Ad-ATP7B-GFP on the copper metabolism of skin fibroblasts of Wilson's disease patients

Abstract

Objective To explore the effect of miniAd-ATP7B-GFP on the copper metabolism of skin fibroblasts of Wilson's disease (WD) patients under high concentration copper medium. Methods Firstly, mini-adenovirus carrier containing human ATP7B gene was built and the mutations of 8 WD patients were detected. Fibroblasts from primary culture of skin of WD patients and normal human were cultivated 72 h in basic medium and medium with the copper concentration of C1 (22.3 μmol/L), C2 (89.2 μmol/L), C3 (156.1 μmol/L), C4 (245.3 μmol/L). Then the concentration of copper and protein was detected and copper/protein ratio was calculated. Secondly, miniAd-GFP (miniAd-GFP group) and miniAd-ATP7B-GFP(miniAd-ATP7B-GFP group) were added into WD patients skin fibroblasts respectively, Wilson non-transfection group and normal group were set up as control, and C4 medium was used to culture the cells of four groups for 72 h and 96 h. Then the concentration of copper and protein was detected and copper/protein ratio was calculated. Results Five kinds of mutations were detected from 8 WD patients. The copper/protein ratio of WD patients and normal human in basic medium and the C1-C3 groups had no statistically significant difference, but in C4 group (WD (1 871.6±209.2) ng/mg, normal group (1 267.2±188.3) ng/mg) the difference was statistically significant (t=6.075, P<0.01). C3 ((816.3±113.9) ng/mg) and C4 groups had statistically significant difference compared with the basic medium group ((159.2±38.6) ng/mg; WD: χ2=31.493, normal group: χ2=30.708, both P<0.01). The copper/protein ratio of 96 h group was higher than 72 h group. Compared with WD non-transfection (96 h: (2 731.2±188.7) ng/mg, 72 h: (1 901.7±219.5) ng/mg) and normal groups, miniAd-ATP7B-GFP group had statistically significant difference both in 96 h ((2 071.0±171.8) ng/mg) and 72 h groups ((1 495.5±161.4) ng/mg; 72 h: F=20.130, 96 h: F=51.496, P<0.01). Conclusion MiniAd-ATP7B-GFP has partial improvement on copper metabolism of skin fibroblasts of WD patients under high concentration copper medium. Key words: Hepatolenticular degeneration; Skin; Fibroblasts; Copper; In vitro techniques