己糖对杜兴肌肉萎缩症核酸药物Pip5e-PMO跳读活性研究

Effect of hexoses on Pip5e-PMO mediated exon-skipping in the Duchenne muscular dystrophy mouse model

目的:在杜兴肌肉萎缩症模型上探究己糖混合物对核酸药物Pip5e-PMO介导的外显子跳读活性的促进作用。方法:选择成年mdx小鼠为测试动物模型,通过局部肌肉和系统尾静脉注射,比较Pip5e-PMO与己糖和生理盐水联用的作用效果,利用免疫荧光(IHC)和蛋白印迹法(Western blot)检测dystrophin阳性肌纤维的数量和分布及dystrophin蛋白的表达水平;利用逆转录-聚合酶链式反应(RT-PCR)检测外显子跳读。结果:在局部肌肉给药试验中,己糖能够促进Pip5e-PMO诱导dystrophin蛋白表达水平(P<0.05)。在系统试验中,对己糖和生理盐水组比较发现:按15 mg/kg的剂量单次系统尾静脉注射Pip5e-PMO,骨骼肌中dystrophin蛋白表达恢复至正常水平的50%;在心脏中,也检测到明显的dystrophin恢复表达和外显子跳读。但两组无显著性差别(P>0.05)。结论: Pip5e-PMO可诱导有效的外显子跳读和dystrophin蛋白表达,但己糖对其促进效果有限。

Objective: To explore the potential of hexose(the mixture of glucose and fructose -GF) in enhancing the delivery of peptide-modified PMO (Pip5e-PMO) in the Duchenne Muscular Dystrophy(DMD) mouse model (mdx mouse). Methods: Pip5e-PMO in GF or saline was intramuscularly or intravenously administered into adult mdx mice. Immunohistochemistry(IHC) and Western blot were used to measure the distribution and expression of dystrophin. Reverse transcription-Polymerase chain reaction (RT-PCR) was utilized to examine the level of exon skipping. Results: Local intramuscular results showed that GF enhanced the activities of Pip5e-PMO demonstrated by the increased number of dystrophin-positive fibers and level of dystrophin expression(P<0.05). Although single intravenous injection of 15mg/kg Pip5e-PMO induced substantial number of dystrophin-positive fibers and therapeutic levels of dystrophin restoration in peripheral muscles and heart of mdx mice treated with either GF or saline, the difference between GF and saline groups was not significant(P>0.05). Conclusion: GF could enhance the activities of Pip5e-PMO intramuscularly but may fail to have any impact on dystrophin-deficient mdx mice.