pcDNA3.0/ATP7B对TX小鼠肝铜代谢影响

Effect of copper metabolism of pcDNA3.0/ATP7B on TX mice

目的探索以重组真核表达质粒pcDNA3.0/ATP7B进行TX小鼠基因治疗的初步疗效。方法以质粒和脂质体混合后进行尾静脉注射,分别给予TX小鼠pcDNA3.0/APT7B 0、10、20、50、100、150μg,3 d后测定其肝铜含量(ng/mg,即每mg干重的肝组织中铜的含量),确定合适的治疗剂量。然后随机选取36只TX小鼠,随机分成3组,即注射生理盐水组、注射pcDNA3.0组、注射pcDNA3.0/ATP7B组。进行肝脏组织RT-PCR、W estern b lot检测和肝铜含量测定。结果根据预实验结果,取pcDNA3.0/APT7B 100μg/只为治疗剂量。RT-PCR和W estern b lot结果显示,加入pcDNA3.0/ATP7B组于加入后第3、7、14天均可见阳性条带。但以加入第3天时最明显。加入生理盐水组和加入pcDNA3.0质粒组均未见阳性条带。加入pcDNA3.0/APT7B组与生理盐水组及pcDNA3.0组比较,于加入第3、7天肝铜含量显著下降(P<0.05),以第3天下降最明显。加入第14天肝铜含量无显著下降,P>0.05。结论ATP7B基因重组真核表达质粒pcDNA3.0/ATP7B对TX小鼠的铜代谢有改善作用。

Objective To explore the impact of copper metabolism of pcDNA3.0/ATP7B on TX mice. Methods pcDNA3. 0/ATPTB was mixed with liposome,and administered into TX mice by tail vein injection. The dosage of pcDNA3.0/ATPTB was 100 p.g each mice. 36 TX mice were selected and divided into 3 groups randomly： group of physiologic saline, group of pcDNA3.0 and group of pcDNA3.0/ATP7B group. On the 3rd, 7th, 14th day, RT-PCR, Western blot, the liver copper index were conducted to investigat whether ATP7 B was expressed and the copper metabolism disturbance was ameliorated in TX mice. Results The human ATP7B proteins were detectable in pcDNA3.0/ ATP7B group,but not in the other two groups. The same results were got in RT-PCR part. On the 3rd,7th day,the liver copper index were significantly ameliorated in pcDNA3.0/ATP7B group than in the other two groups. On14th day,the liver copper index was not significantly decreased in pcDNA3.0/ATP7B group. Conclusion pcDNA3.0/ATP7B could ameliorate the copper metabolism disturbance in TX mice.