Fusigenic liposome-mediated DNA transfer into cardiac myocytes.
Fusigenic liposome-mediated DNA transfer into cardiac myocytes.
复制标题
融合脂质体介导的 DNA 转移到心肌细胞中。
DOI：
10.1006/jmcc.1996.0130
复制
发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Dzau,VJ
中科院分区：
文献类型：
--
作者：
Ellison,KE;Bishopric,NH;Webster,KA;Morishita,R;Gibbons,GH;Kaneda,Y;Sato,B;Dzau,VJ
目前将基因转移到培养的心肌细胞中的方法具有严重的局限性，包括低效率、毒性或对DNA插入片段大小的限制。本研究探讨了日本血凝病毒（HVJ）在促进脂质体介导的DNA转移到培养的新生大鼠心肌细胞的有效性。将异硫氰酸黄绿素标记的寡核苷酸（F-ODN）或编码SV 40大T抗原（pActSVT）和β-半乳糖苷酶（pActβ-gal）的质粒表达载体与脂质体和HVJ的病毒蛋白外壳复合。在HVJ-脂质体包封之前，将质粒载体与核定位蛋白HMG-1复合。通过在培养第3天或第7天与HVJ-脂质体/DNA复合物孵育转染新生肌细胞。使用F-ODN，我们能够证明转染后1小时DNA的显著摄取（转染效率为80-90%），其在培养物中持续1周。有趣的是，F-ODN在转染后的前4天集中在肌细胞核中。免疫组化结果显示，无论是在培养的第3天还是第7天，转染pActSVT或pActβ-Gal的心肌细胞在72 h时都有25-30%表达质粒编码的蛋白，而转染空白载体的细胞则没有。定量β-半乳糖苷酶测定证实HVJ的使用显著增强脂质体介导的转染。细胞毒性不明显。通过冠状动脉内注射的基因转移也证明了HVJ介导体内兔心肌细胞转染的能力，F-ODN依赖性荧光持续长达1周。结论HVJ/脂质体介导的寡核苷酸和质粒转染心肌细胞的方法在体外和体内均有效，为心肌细胞生物学和疾病的研究提供了新的工具。
Current methods of gene transfer into cultured cardiac myocytes have serious limitations, including low efficiency, toxicity or constraints on DNA insert size. The present study examined the effectiveness of hemagglutinating virus of Japan (HVJ) in promoting liposome-mediated DNA transfer into cultured neonatal rat cardiac myocytes. Fluorescein isothiocyanate-labeled oligonucleotides (F-ODN) or plasmid expression vectors encoding SV40 large T antigen (pActSVT) andβ-galactosidase (pActβ-gal) were complexed with liposomes and the viral protein coat of HVJ. Plasmid vectors were complexed with the nuclear localizing protein HMG-1 prior to HVJ-liposome encapsulation. Neonatal myocytes were transfected by incubation with HVJ-liposome/DNA complexes on culture day 3 or 7. Using F-ODN, we were able to demonstrate significant uptake of DNA (transfection efficiencies of 80–90%) 1 h after transfection that persisted for 1 week in culture. Interestingly, F-ODN were concentrated in the myocyte nuclei for the first 4 days after transfection. Immunohistochemistry showed that 25–30% of myocytes transfected with either pActSVT or pActβ-Gal expressed plasmid-encoded protein at 72 h whether they were transfected at day 3 or day 7 of culture, while cells transfected with blank vectors did not. Quantitativeβ-galactosidase assays confirmed that the use of HVJ significantly enhanced liposome-mediated transfection. Cell toxicity was not apparent. Gene transfer via intracoronary injection also demonstrated the capacity of HVJ to mediate transfection of rabbit cardiac myocytesin vivo, with F-ODN-dependent fluorescence persisting for up to 1 week. We conclude that HVJ/liposome-mediated transfer is efficient for the transfection of both oligonucleotides and plasmids into cardiac myocytes bothin vitroandin vivo, and may provide a new tool for the investigation of cardiac myocyte biology and disease.