【摘要】 非病毒载体介导的外源基因在哺乳动物骨骼肌细胞中的表达往往受限于基因转移效率的低下.本文利用电穿孔为基因转移方法,研究了人对氧磷酶基因(PON1)在原代培养的小鼠骨骼肌成肌细胞和成熟肌管中的转移与表达.在上述细胞中加入PON1的真核表达质粒后实施一定条件的电穿孔,通过测定不同时间点培养基与细胞裂解液中芳香酯酶活性的变化以衡量PON1的表达与分泌.结果显示,PON1在成肌细胞中表达的最佳电穿孔条件为800 V/cm,20 ms and 50μF;在肌管中为700 V/cm,20 ms and 50μF.在此条件下,细胞存活率均达75%以上,且表达的蛋白均可有效分泌.RT-PCR分析同样验证了PON1 mRNA在骨骼肌细胞中的高效表达.电穿孔介导的PON1基因表达效率显著高于传统的基因转移方法如磷酸钙法和阳离子脂质体法.因此,以不同分化阶段的骨骼肌细胞为靶细胞,通过电穿孔介导外源基因表达切实可行,并可能在细胞工程与基因治疗等领域均具有潜在的应用前景.更多还原

【Abstract】 Gene transfer in primary mammalian myocytes via non-viral methods has been cumbered by the low efficiency of transfection.We developed a simple and reproducible procedure to transfect human paraoxinase 1 gene(PON1) into the primary cultures of mouse myoblasts and mature muscle cells.Electroporation was used to promote the PON1 delivery in mouse myogenic cells and myotubes with high cell viability.The highest arylesterase activities in culture medium of myoblasts was 0.954±0.187(U/ml) under the conditions of 800(V/cm),20 ms and 50 μF,whereas in myotubes,it was 2.611 ± 0.420(U/ml) under 700(V/cm),20 ms and 50 μF.Respectively 87% and 84% of the expressed PON1 in myoblasts and myotubes were secreted into culture medium after electroporations.The PON1 mRNA in mouse myocytes was detectable by RT-PCR.As the efficiency of PON1 expression in muscle cells with our approach was significantly higher than that by calcium phosphate or cation lipid method,it might be potentially useful for the non-viral delivery in bioengineering and gene therapy.更多还原