Effect Of Mini Ad-ATP7B-GFP On The Copper Metabolism Of Skin Fibroblasts Of Wilson’s Disease Patients

Background Wilson’s disease(WD) is also called hepatolenticular degeneration, which is a autosomal recessive inherited copper metabolic disorder caused by ATP7B gene mutation. So far, the therapeutic methods of WD include:removing copper treatment of metal complexing agent, Zinc treatment and liver transplantation. Metal complexing agent such as penicillamine is a symptomatic treatment, which has certain curative effect for early stage of Wilson’s disease. After treatment, Some patients get back to normal work and life. However, patients must be lifelong medication. Additionly, it accompany great minus effects, up to 70% patients experienced the side effects and some patients can’t hold on. Liver transplantation therapy only can remission part of patients because of its strong immune rejection, very limited cadaver organs and expensive expense. So people begin to explore wilson’s disease gene therapy gradually because of the imitations of traditional treatment described above. Wilson’s disease is a single gene genetic disease, gene therapy is the most fundamental treatment.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 22.30 (C1),89.20 (C2),156.1 (C3),245.3 μmol/L (C4). 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 fibroblasts skin 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 (1871.6 ±209.2) ng/mg, normal group (1267.2± 188.3) ng/mg) the difference was statistically significant (t=6.075, P<0.01). C3 and C4 groups had statistically significant difference compared with the basic medium group (WD:x2=31.493, normal group:x2=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 and normal groups, miniAd-ATP7B-GFP group had statistically significant difference both in 96 h and 72 h group (72 h:F=20.130,96 h:F=51.496, both P<0.01).Conclusion MiniAd-ATP7B-GFP has partial improvement on copper metabolism of skin fibroblasts of WD patients under high concentration copper medium.