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聚乙烯亚胺增强Pluronic介导的BALB/c小鼠骨骼肌原位基因传递和表达效率
Polyethyleneimine Enhances in situ Gene Delivery and Expression in Skeletal Muscles of BALB/c Mice in the Pluronic-Mediated System
何雨桐, 孙哲, 王刚*
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DOI:10.11984/j.issn.1000-7083.20180156
作者单位:四川大学生物材料工程研究中心, 成都 610064
中文关键字:BALB/c小鼠;骨骼肌基因传递;聚乙烯亚胺;Pluronic L64
英文关键字:BALB/c mice; skeletal muscle gene delivery; polyethyleneimine; Pluronic L64
中文摘要:目的 在Pluronic L64介导的BALB/c小鼠骨骼肌基因传递体系中正确引入聚乙烯亚胺(PEI)以增强基因表达效率。方法 以生理盐水组、质粒DNA(pDNA)组和Pluronic L64/pDNA(L/D)组为对照组,各比例PEI/pDNA与Pluronic L64混合物(L/P/D-0.5、L/P/D-3、L/P/D-10)为实验组,将各组样品注射到小鼠胫骨前肌中。7 d后取组织,检测报告基因表达(β-半乳糖苷酶和荧光素酶)和肌肉组织病变情况。通过尾静脉注射考察L/P/D-0.5组的体内急性毒性。最后,通过动态光散射仪和原子力显微镜测定L/P/D-0.5组中PEI/pDNA复合物的粒径、电位及形貌。结果 1)报告基因表达评估:L/P/D-0.5组外源基因表达效率最高;随着N/P增加,几乎检测不到外源基因的表达。2)生物相容性评估:无论原位注射还是尾静脉注射,L/P/D-0.5组均没有显示出组织病变,而其他比例的L/P/D组出现了不同程度的组织学病变。3)物理特征表征:虽然在L/P/D-0.5组中DNA未被完全压缩,但PEI/pDNA复合物已拥有良好的纳米粒径和形态结构,且表面电位为负。结论 L/P/D-0.5体系能安全有效地增强外源基因在骨骼肌内的表达。
英文摘要:Objective To correctly introduce cationic polyethyleneimine (PEI) into the Pluronic L64-mediated gene delivery system of mice skeletal muscle to enhance the efficiency of gene expression.Methods In this study, different PEI samples were transferred into mice tibialis anterior muscles via intramuscular injection. The saline, plasmid DNA (pDNA) and Pluronic L64/pDNA (L/D) were used as control groups, and the mixtures of PEI/pDNA with different N/P ratios (L/P/D-0.5, L/P/D-3, and L/P/D-10) and Pluronic L64 were used as experimental groups. Muscle tissues were taken out at 7 days after injection, and then the report gene expression of β-galactosidase and luciferase and the potential pathological changes of muscle tissues were measured. In addition, the in vivo acute toxicity experiment was further determined by injecting L/P/D-0.5 through tail vein. Finally, the particle size, zeta potential and morphology of PEI/pDNA complexes in L/P/D-0.5 system were detected by dynamic light scattering instrument and atomic force microscope (AFM).Results 1) The L/P/D-0.5 group showed the highest report gene expression; As N/P ratio increasing, the report gene expression could hardly be detected. 2) The result of biocompatibility assessment showed that no tissue lesion was observed in L/P/D-0.5 group irrespective of muscular injection or intravenous injection, while other ratios of L/P/D showed histologic lesions with varying degrees. 3) PEI/pDNA complexes were spherical shape with nanometer size, and this might contribute to the process of gene delivery in skeletal muscle-based system, although pDNA was not fully compressed in L/P/D-0.5 group.Conclusion The L/P/D-0.5 system can safely and effectively enhance the expression of exogenous genes in skeletal muscle.
2018,37(6): 620-627 收稿日期:2018-03-14
分类号:Q95-336
基金项目:国家自然科学基金项目(31370972)
作者简介:何雨桐(1992—),女,硕士研究生,研究方向:生物化学与分子生物学,E-mail:kelseyhe@foxmail.com
*通信作者:王刚,研究员,研究方向:靶向基因传递、基因治疗,E-mail:wgang@scu.edu.cn
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