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目的探讨多价阳离子脂质体介导的pCKM-mPTH质粒治疗甲状旁腺功能低下症(HPT)的最佳方案。方法用DNaseⅠ敏感性试验测定脂质体对质粒的保护作用,用不同比例的脂质复合物及裸质粒进行体内外转染,收集细胞培养液和血清,采用放射免疫法测定其甲状旁腺激素(PTH)含量。结果脂质复合物比例为2∶1时,即对DNA有明显的保护作用;在体外转染实验中,复合物比例在3∶1时PTH表达量最高,为每24小时(16.4±4.8)pg/10 000细胞,而在此比例下pCKM-mPTH质粒达20μg/100μl时,模型体内PTH的血清浓度最高,为35.9 pg/m l±2.2 pg/m l。结论与裸质粒相比,多价阳离子脂质体更适宜于作为HPT基因治疗的非病毒载体,而其介导重组质粒pCKM-mPTH直接转染肌肉的良好效能取决于脂质复合物的构成比例和剂量。
OBJECTIVE: To investigate the optimal treatment of hypoparathyroidism (HPT) by polyvalent cationic liposome-mediated plasmid pCKM-mPTH. Methods The protective effect of liposomes on plasmid was determined by DNase Ⅰ sensitivity assay. In vitro and in vivo transfection were performed with different ratios of lipid complexes and naked plasmid, and the cell culture medium and serum were collected. The levels of parathyroid hormone (PTH) content. RESULTS: The ratio of lipoplexes was 2: 1, which indicated that the DNA complex had a significant protective effect on DNA. In vitro transfection experiments, the highest PTH expression was at 3: 1, which was 16.4 ± 4.8 pg / 10 000 cells, while the pCKM-mPTH plasmid in this ratio up to 20μg / 100μl, the model serum PTH highest concentration of 35.9 pg / ml ± 2.2 pg / ml. Conclusions Multivalent cationic liposomes are more suitable as non-viral vectors for HPT gene therapy than naked plasmids and their good potency for direct transfection of the recombinant plasmid pCKM-mPTH into muscle depends on the compositional ratio of the lipid complex And dose.